Peptides targeting TNF family receptors and antagonizing TNF action, compositions, methods and uses thereof

ABSTRACT

The present invention provides modulators of TNF, particularly peptides and their derivatives, particularly GEP peptides, which antagonize TNF and TNF-mediated responses, activity or signaling. The invention provides methods of antagonizing TNF and the modulation of TNF-mediated diseases or responses, including inflammatory diseases and conditions. Compositions of GEP peptides, including in combination with other inflammatory mediators, are provided. Methods of treatment, alleviation, or prevention of TNF-mediated diseases and inflammatory conditions, including rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, psoriasis, inflammatory bowel diseases, Chrohn&#39;s disease, ulcerative colitis, uveitis, inflammatory lung diseases, chronic obstructive pulmonary disease, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional application claiming thepriority of copending provisional application Ser. No. 61/212,971, filedApr. 17, 2009, the disclosure of which is incorporated by referenceherein in its entirety. Applicants claim the benefits of thisapplication under 35 U.S.C. §119 (e).

GOVERNMENTAL SUPPORT

This invention was made with government support under NIH/NIAMS1 K01AR053210 and NIH/NIA 1 R03 AG029388, awarded by the National Instituteof Health, National Institute of Arthritis and Musculoskeletal and SkinDiseases. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to modulators of TNF/TNFR,particularly peptides and their derivatives which antagonize TNF andTNF/TNFR-mediated responses, activity or signaling. The invention alsorelates to methods of antagonizing TNF and the modulation ofTNF-mediated diseases or responses, including inflammatory diseases andconditions.

BACKGROUND OF THE INVENTION

In the progress of arthritis, synovium, cartilage, and bone are allsites of increased production of growth factors, cytokines, andinflammatory mediators that are believed to contribute to pathogenesis[1, 2]. Although both bone and synovium have important roles in thepathogenesis of arthritis [1, 3], most efforts at developingdisease-modifying treatments have focused on the molecular events withincartilage. Arthritic chondrocytes undergo a series of complex changes,including proliferation, catabolic alteration, and, ultimately, death.The regulation of these phenotypic changes at different stages ofdisease is under intensive study, with focus on the biomechanical andbiochemical signals that regulate each of these discrete chondrocyteresponses [2, 4]. Chondrocytes themselves are major protagonists in thisregulatory cascade—not just the target of external biomechanical andbiochemical stimuli, but themselves the source of cytokines, proteases,and inflammatory mediators that promote the deterioration of articularcartilage [1, 2]. Pathogenic molecules produced by arthriticchondrocytes include tumor necrosis factor (TNF), interleukin-1 (IL-1),IL-6, IL-8, matrix metalloproteinases (MMPs), ADAMTSs, nitric oxide,prostaglandins, and leukotrienes [2, 4]. There is also evidence thatarthritic chondrocytes exhibit increased anabolic activity, includingincreased release of growth factors and synthesis of type II collagen,proteoglycan, and other extracellular matrix proteins, as well as theexpression of genes associated with the chondroprogenitor hypertrophicphenotype [5-7].

A great deal of research in rheumatology over the past two decades hasfocused on identifying cytokines and mediators responsible for theinflammatory and degenerative processes in rheumatoid arthritis (RA),with the aim of developing specific antagonists of therapeutic value.Among all factors, TNF-a has received the greatest attention because ofits position at the apex of the pro-inflammatory cytokine cascade, andits dominance in the pathogenesis of RA. Many lines of evidence supportthis theory including: (1) TNF-a is expressed at high levels in inflamedsynovium and cartilage from RA patients; (2) anti-TNF-a inhibits theproduction of other pro-inflammatory cytokines including IL-1; and (3)TNF-a can induce joint inflammation, trigger cartilage destruction byinducing metalloproteinase, and stimulate osteoclastogenesis and boneresorption. Most importantly, anti-TNF therapies for RA have shownremarkable results by decreasing inflammation, improving patientfunction and vitality, and attenuating cartilage and bone erosions.There are now three anti-TNF treatments via targeting to TNF ligand,etanercept (Enbrel, a soluble TNFR2-IgG1 fusion protein), infliximab(Remicade, a chimeric monoclonal antibody against TNF-a), and adalimumab(a humaneric monoclonal antibody against TNF-a) that have been usedclinically for treating various kinds of inflammatory diseases,including rheumatoid arthritis. Engineered proteins/peptides are nowproviding a new wave of therapeutic products. Indeed, designedprotein/peptide therapeutic agents now outnumber and surpass the numberof new small-molecule drugs approved annually by the FDA. Antibodies andimmunoadhesins that directly target cytokines for their systemic removal(ligand ablation) have become an effective therapeutic strategy (e.g.etanercept, adalimumab and infliximab), and in some indications theselective targeting of cytokine receptors (e.g. anakinra) can deliver ahighly effective clinical outcome.

Granulin/epithelin precursor (GEP), also known as PC-cell-derived growthfactor (PCDGF), acrogranin, progranulin (PGRN), proepithelin (PEPI), orGP80, was first purified as a growth factor from conditioned tissueculture media [8, 9, 65, 66, 67]. GEP is a 593-amino-acid secretedglycoprotein with an apparent molecular weight of 80 kDa [10, 14], whichacts as an autocrine growth factor. GEP contains seven and a halfrepeats of a cysteine-rich motif (CX₅₋₆CX₅CCX₈CCX₆CCXDX₂HCCPX₄CX₅₋₆C)(SEQ ID NO: 9) in the order P-G-F-B-A-C-D-E, where A-G are full repeatsand P is the half motif (FIG. 1). The C-terminal region of the consensussequence contains the conserved sequence CCXDX₂HCCP (SEQ ID NO: 10) andis suggested to have a metal binding site and to be involved inregulatory function [15]. Notably, GEP undergoes proteolytic processingwith the liberation of small, 6-kDa repeat units known as granulins (orepithelins), which retain biological activity [16]: peptides are activein cell growth assays [13] and may be related to inflammation [17].

GEP is abundantly expressed in rapidly cycling epithelial cells, incells of the immune system, and in neurons [10-12, 17]. High levels ofGEP expression are also found in several human cancers and contribute totumorigenesis in diverse cancers, including breast cancer, clear cellrenal carcinoma, invasive ovarian carcinoma, glioblastoma, adipocyticteratoma, and multiple myeloma [16, 18-24]. Although GEP mainlyfunctions as a secreted growth factor, it was also found to be localizedinside cells and to directly modulate intracellular activities [12,25-27]. The role of GEP in the regulation of cellular proliferation hasbeen well characterized using mouse embryo fibroblasts derived from micewith a targeted deletion of the insulin-like growth factor receptor(IGF-IR) gene (R-cells). These cells are unable to proliferate inresponse to IGF-1 and other growth factors (EGF and PDGF) necessary forprogression through the cell cycle [28]. In contrast, GEP is the onlyknown growth factor able to bypass the requirement for the IGF-IR, thuspromoting cell growth of R-cells [13, 29]. Increasing evidence has alsoimplicated GEP in the regulation of differentiation, development andpathological processes. It has been isolated as adifferentially-expressed gene from mesothelial differentiation [30],sexual differentiation of the brain [31], macrophage development [32],and synovium of rheumatoid arthritis and osteoarthritis [33]. GEP wasalso shown to be a crucial mediator of wound response and tissue repair[21, 34]. It was reported that mutations in GEP cause tau-negativefrontotemporal dementia linked to chromosome 17 [35-38]. The mode ofaction of GEP remains largely unknown. Several GEP-associated proteinshave been reported to affect GEP action in various processes. Oneexample is the secretory leukocyte protease inhibitor (SLPI). Elastasedigests GEP exclusively in the intergranulin linkers, resulting in thegeneration of granulin peptides, suggesting that this protease may be animportant GEP convertase. SLPI blocks this proteolysis either bydirectly binding to elastase or by sequestering granulin peptides fromthe enzyme [34]. GEP can modulate transcriptional activities byinteracting with human cyclin T1 [26] and Tat-P-TEFb [25]. GEP was alsofound to interact with perlecan, a heparan sulfate proteoglycan;perlecan-null mice exhibit severe skeletal defects [19, 39-41].

The Tumor Necrosis Factor (TNF) family of cytokines plays an essentialrole in multiple biological functions including inflammation,organogenesis, host defense, autoimmunity, and apoptosis. The action ofthese potent biological mediators is achieved through a receptor-ligandinteraction, leading to intracellular signaling and a change in cellularphenotype. The ligands exert their function by forming trimers andbinding to their corresponding receptors. Subsequent receptoroligomerization results in conformational change of the receptor'sintracellular domain, which then allows for members of the TNFreceptor-associated factor (TRAF) family of adaptor proteins to bind andinitiate a signaling cascade. TNFR2, TNFR1, TrkA, NGFR, CD 40, CD 30,OX-40, DR5, DR3, DR4 and RANK include some of the members of TNFreceptor super-family that interact with different TRAF molecules(including 1-6) (Lewit-Bentley, A., et al., J. Mol. Biol. 199:389-392(1988), Banner, D. W., et al., Cel. 73:431-445 (1993), Karpusas, M., etal., Structure. 3:1031-1039 (1995), Hymowitz, S. G., et al., Mol. Cell.4:563-571 (1999), Mongkilsapaya, J., et al., Nat. Struc. Biol.6:-1048-1053 (1999), Cha, S. S., et al., J. Biol. Chem. 275:31171-31177(2000)).

Both TNF receptors (TNFR1 and TNFR2) are ubiquitously expressed in cellsand interact with their cognate ligand: TNFα, a central proinflammatorycytokine [42-44]. It is widely accepted that TNFα serves very importantfunctions in pathophysiology, being a factor that interferes stronglywith the cell growth, differentiation and death. TNF appears not only toorchestrate acute responses to infection and immunological injury butalso to act as a balancing factor required for the re-establishment ofphysiological homeostasis and regulation [45]. TNFα has been found toaffect skeletal development: its level is increased in most inflammatorydiseases known to affect longitudinal growth in children [46, 47] andcatch-up growth was shown in children with refractory juvenileidiopathic arthritis treated with the TNF antagonist etanercept (Enbrel)[46, 47]; TNFα regulates growth plate chondrocytes and suppresslongitudinal growth in metatarsal organ cultures [48].

Arthritis is a degenerative joint disease, occurring primarily in thesenior population, that currently affects more than 46,000,000individuals in the United States. Typical clinical symptoms are pain andstiffness, particularly after prolonged activity. In industrializedsocieties arthritis is the leading cause of physical disability,increased health care usage, and impaired quality of life. The impact ofarthritic conditions is expected to grow as the population bothincreases and ages in the coming decades. Despite the prevalence ofarthritic diseases, their precise etiology, pathogenesis, andprogression remain beyond our understanding. Evidence is accumulatingthat demonstrates the significance of inflammatory cytokines and growthfactors in the pathological processes of arthritis. The destruction ofthe extracellular matrices of articular cartilage and bone in arthriticjoints is thought to be mediated by excessive cytokine activities andimbalance between inflammatory cytokines and their physiologicalantagonists. The isolation of the growth factors that regulateschondrocytes and arthritis, and the inhibitors that antagonize theactions of cytokines, are therefore of great importance from both apathophysiological and a therapeutic standpoint. We have previouslyidentified granulin/epithelin precursor (GEP) as a novel chondrogenicgrowth factor that plays an essential role in cartilage formation (Xu, Ket al (2007) J Biol Chem 282(15):11347-11355; WO 2008/094687 A2).

There still exists a need in the art for a better and more completeunderstanding of the process of and, thereby, intervention for,inflammatory diseases and conditions, particularly TNF family membermediated processes and conditions. Thus, the purpose of this inventionis to extend our understanding of the molecular mechanisms by whichgrowth factors and cytokines control cartilage development andarthritis, and to mediators thereof for development of new anti-TNF/TNFRtherapeutic interventions for various kinds of TNF-related diseases,including inflammatory arthritis.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

Taking into account the biological properties of GEP, it has beenhypothesized that GEP could act through “classic” membrane receptor(s),as do other known growth factors. Thus far, a functional receptor hasnot been identified. Our functional genetic screen described herein ledto the isolation of TNF receptors as novel GEP-binding receptors. Ourstudies demonstrate that GEP (Granulin/epithelin precursor) is the firstgrowth factor that directly targets to TNF receptors (TNFR). Thus GEPand its derived peptide(s) represent a novel anti-TNF/TNFR signalingblocker by acting on the cytokine receptors.

The studies described herein demonstrate that GEP is a novel antagonistof TNF/TNFR signaling. The present findings reveal that: 1) GEP directlybinds to TNF receptors in a dose-dependent manner; 2) Blocking TNFreceptors by neutralizing antibodies or recombinant extracellulardomains abolishes GEP function in stimulation of cell proliferation; 3)GEP potently activates Erk1/2 signaling, and moderately Akt pathway; 4)GEP activates genes known to be the downstream molecules of the TGFβsubfamily, including BMP2; in addition, GEP-mediated inductions of thesegene expressions depend on TNF receptors; 5) GEP is anarthritis-responsive gene and its level was significantly elevated inpatients with arthritis; 6) GEP, as an antagonist of TNFα, dramaticallyreduces inflammation response and apoptosis induced by TNFα; and 7)importantly, GEP exhibits better (at least as good as) inhibition ofTNF-stimulated inflammation than Enbrel and Remicade that have been usedclinically for treating various kinds of inflammatory diseases andconditions, including rheumatoid arthritis. These findings reveal thatGEP is a novel naturally-occurring antagonist of TNF/TNFR signaling viadirectly targeting to TNF receptors.

The invention provides GEP and GEP peptides, particularly including thepeptide(s) denoted atsttrin, as modulators of TNF/TNFR activity andsignaling, particularly inhibiting or blocking TNF-mediated signaling orresponse, particularly as antagonists of TNF/TNFR.

The invention provides peptides which antagonize TNF family memberreceptors, particularly TNFR, and block, inhibit, reduce, or prevent TNFfamily member signaling, including TNF/TNFR signaling. The inventionprovides peptides which antagonize TNF family member receptors,particularly RANK, and block, inhibit, reduce, or prevent TNF familymember signaling, including RANK/RANKL signaling.

In a particular embodiment, the present invention relates to all membersof the herein disclosed family of GEP peptides and of atsttrin, whichare capable of modulating, particularly antagonizing, TNFfamily/receptor signaling and response, particularly TNF/TNFR signalingand response. The family of peptides includes fragments or portions,including mixed portions of GEP sequence and half units, particularlycomprising one or more granulin unit and one or more linker unit of GEP.In one aspect the peptide comprises two or more half units of granulinunits and one or more linker unit of GEP.

In a particular aspect of the invention, the GEP peptide comprises thepeptide atsttrin, comprising combinations of half units of granulinunits A, C and F in combination with linker units P3, P4 and P5. In aparticular aspect, the GEP peptide comprises a combination of half unitsof granulin units, wherein at least one half unit is ½F, and linkerunits, particularly at least two linker units. In a further particularaspect atsttrin has the amino acid sequence set out in FIG. 24 andcomprises granulin units and linker units ½F-P3-P4-½A-P5-½C, includingas set out in SEQ ID NO: 2.

The present invention naturally contemplates several means forpreparation of the GEP peptides and/or atsttrin of the presentinvention, including as illustrated herein and/or using knownrecombinant techniques, and the invention is accordingly intended tocover such synthetic preparations within its scope. The determination ofthe antagonist amino acid sequences disclosed herein facilitates thereproduction of the peptides by any of various synthetic methods or anyknown recombinant techniques, and accordingly, the invention extends toexpression vectors comprising nucleic acid encoding the peptides of thepresent invention for expression in host systems by recombinant DNAtechniques, and to the resulting transformed hosts.

The present invention also relates to a recombinant DNA molecule,recombinant nucleic acid, or cloned gene, or a degenerate variantthereof, preferably a nucleic acid molecule, in particular a recombinantDNA molecule or cloned gene, encoding the amino acid of one or more GEPpeptides shown in FIG. 24 or variants thereof. In a particularembodiment, the recombinant DNA molecule, recombinant nucleic acid, or adegenerate variant thereof, preferably a nucleic acid molecule, encodesa GEP peptide capable of antagonizing TNF/TNFR, which comprises one ormore granulin unit and one or more linker unit of GEP as depicted inFIG. 1 and as set out in the sequence of GEP of FIG. 23. In a furtherparticular embodiment, the recombinant DNA molecule, recombinant nucleicacid, or a degenerate variant thereof, preferably a nucleic acidmolecule, encodes a GEP peptide atsttrin capable of antagonizingTNF/TNFR as set out in FIG. 24 and comprising granulin units and linkerunits ½F-P3-P4-½A-P5-½C (SEQ ID NO: 2).

It is an object of the present invention to provide pharmaceuticalcompositions for use in therapeutic methods which comprise or are basedupon the GEP peptides and/or atsttrin. The pharmaceutical compositionsinclude combinations of one or more GEP peptides and/or atsttrin havingTNF antagonistic activity. The pharmaceutical compositions includecombinations of one or more GEP peptides and/or atsttrin having TNFantagonistic activity and one or more inflammatory mediator.Inflammatory mediators include and may be selected from non-steroidalanti-inflammatory agents (NSAIDs), steroids, corticosteroids, other TNFantagonists (e.g. etanercept, adalimumab and infliximab), and cytokinereceptor antagonists (e.g. anakinra). The pharmaceutical compositionsinclude combinations of one or more GEP peptides and/or atsttrin havingTNF antagonistic activity and one or more inflammatory mediator,immunododulatory agent, or anti-cancer agent.

In a further embodiment, the present invention relates to certaintherapeutic methods which would be based upon the TNF/TNFR antagonisticactivity of GEP, GEP peptides and/or atsttrin, or active fragmentsthereof, or upon agents or other drugs determined to possess the sameactivity. Thus, the present invention provides methods of preventingand/or treating diseases mediated by TNF/TNFR activity and/or which arefacilitated or induced by TNF family ligand/receptor activity and/orcharacterized by inflammation. The invention provides methods oftreatment, alleviation, or prevention of TNF-mediated diseases andinflammatory conditions or immunological conditions, includingrheumatoid arthritis, osteoarthritis, ankylosing spondylitis, psoriasis,inflammatory bowel diseases, Chrohn's disease, ulcerative colitis,uveitis, inflammatory lung diseases, chronic obstructive pulmonarydisease.

In one aspect, the invention provides a method for treatment,alleviation or prevention of tumors or cancer which are mediated by GEP,TNF, GEP/TNFR and/or TNF/TNFR. Thus, it is contemplated that a tumor,cancerous or precancerous condition wherein the growth and/orproliferation of the tumor or cancerous/precancerous cells are dependenton or facilitated by GEP, TNF, GEP/TNFR and/or TNF/TNFR activity orsignaling may be sensitive to the TNF/GEP antagonist peptide(s) of thepresent invention. The invention further contemplates use andapplication of GEP peptides, particularly including atsttrin to inhibitor block GEP-mediated cancer or cell proliferation.

More specifically, the therapeutic method provides for methods for thetreatment, prevention or alleviation of diseases mediated by TNF/TNFRactivity and/or which are facilitated or induced by TNF familyligand/receptor activity and/or characterized by inflammation by theadministration of pharmaceutical compositions that comprise effectiveantagonists of TNF based on GEP, GEP peptides and/or atsttrin or itssubunits, or other equally effective drugs developed for instance by adrug screening assay prepared and used in accordance with a furtheraspect of the present invention. In a particular aspect, GEP, GEPpeptides, and/or atsttrin may be administered to treat, alleviate, orprevent a TNF/TNFR mediated disease or condition or an inflammatorydisease or condition.

In a further aspect, GEP, GEP peptides and/or atsttrin or its subunitsmay be utilized in methods to modulate, prevent, treat or alleviateimmunological injuries, including allergies, auto-immune diseases andother such immune conditions, particularly wherein TNF/TNFR is involvedor implicated. Thus, immune and/or inflammatory responses inauto-immune, allergies or other such immunological conditions may bemodulated by GEP, GEP peptides and/or atsttrin or its subunits. In onesuch aspect, auto-immune diseases, such as lupus and multiple sclerosis,may be modulated, alleviated or treated by GEP, GEP peptides and/oratsttrin or its subunits.

In particular, the GEP, GEP peptides, atsttrin peptides of the presentinvention, including as described herein and provided in FIGS. 23 and 24herein, their antibodies, agonists, mimics, or active fragments thereof,could be prepared in pharmaceutical formulations for administration ininstances wherein anti-TNF or TNF/TNFR family antagonist activity and/ortherapy is appropriate, such as to treat or alleviate a TNF/TNFRmediated condition or inflammation. The GEP, GEP peptides and atsttrinpeptides include exemplary GEP as set out in SEQ ID NO: 1 and 8, and GEPpeptides or atsttrin peptides as set out in SEQ ID NO: 2 and 3-7, andvariants or subunits thereof. The specificity of the GEP peptides and/oratsttrin hereof would make it possible to better manage the aftereffectsof current anti-TNF and/or anti-inflammatory therapy, and would therebymake it possible to apply broadly as a general anti-TNF and/or anti-TNFfamily agent.

The invention includes an assay system for screening of potential drugsor compounds effective to modulate TNF/TNFR activity of target mammaliancells by mimicking the activity of the GEP peptides. This aspectincludes assays to screen for additional active GEP fragments,granulin/linker unit combinations, derivatives, variants and amino acidmodifications effective to modulate TNF/TNFR activity in a like mannerto GEP and atsttrin peptide. In one instance, the test drug or compoundis administered to a cellular sample with TNFα to activate TNF/TNFRactivity, to determine the effect of the test drug or compound upon TNFαactivity, by comparison with a control, including wherein the control isGEP, active GEP peptide(s), atsttrin.

In an assay, a control quantity of the GEP, GEP peptides, atsttrin,TNFα, TNFR, or antibodies thereto, or the like may be prepared andlabeled with an enzyme, a specific binding partner and/or a radioactiveelement, and may then be introduced into a cellular sample. After thelabeled material or its binding partner(s) has had an opportunity toreact with sites within the sample, the resulting mass may be examinedby known techniques, which may vary with the nature of the labelattached. In the instance where a radioactive label, such as theisotopes ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I,¹³¹I, and ¹⁸⁶Re are used, known currently available counting proceduresmay be utilized. In the instance where the label is an enzyme, detectionmay be accomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric or gasometrictechniques known in the art.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of GEP growth factor. For the unitconsensus sequence C represents cysteine, D aspartic acid, P proline, Tthreonine, G glycine, H histidine; dots represent any amino acids.

FIG. 2 (A) Binding of GEP to TNFR in Yeast. Each Pair of plasmids, asindicated, was co-transformed into yeast strain MAV203. Yeasttransformants were selected on SD-leu⁻/trp⁻/his⁻/ura⁻/3AT⁺ plates andtested for β-galactosidase activity. The known interaction between c-Junand c-Fos was used as a positive control, whereas the lack ofinteraction between Rb and lamin was used as a negative control. (B) GEPassociates with TNFR2 in chondrocytes. Cell extracts prepared from humanchondrocytes were incubated with control IgG, anti-TNFR2 antibodiesfollowed by protein A-agarose. The immunoprecipitated protein complexand cell extracts (lane 1, a positive control) were examined byimmunoblotting with anti-GEP antibodies.

FIG. 3 depicts solid phase binding of TNFR1 extracellular domain(TNFR1ECD) and TNFR2 extracellular domain (TNFR2ECD) to recombinant GEPcoated on microtiter plates.

FIG. 4 (A) Schematic diagram of TNFR2 constructs used to map those ofits fragments that bind to GEP. (B) β-Galactosidase assays.

FIG. 5 depicts an MTT assay. Human chondrocytes were cultured in theabsence (CTR) or presence of 50 ng/ml GEP (GEP) or GEP plus either 1ug/ml of anti-TNFR1 (GEP+TNFR1 ab), anti-TNFR2 (GEP+TNFR2 ab) or 1 ug/mlof anti-IGF1R (GEP+TGF1R ab, employed as a control), and cellproliferation was analyzed using an MTT assay.

FIG. 6. GEP activates Akt and Erk1/2 pathways in human C28I2chondrocytes. Note both long-time (5 min, left panel) and short-time (30sec. right panel) exposures of the film are presented.

FIG. 7 depicts expression profiling of the genes Gadd45b, JunB, KLF2,Smad7, Sox4 and Tcf8 using real-time PCR with primary wild type (B6),TNFR1−/−, and TNFR2−/− MLE cells cultured in the presence of 50 ng/mlGEP for various time points.

FIG. 8 depicts a model for illustrating intracellular events, includingsignaling and target gene expression.

FIG. 9 (Left) Schematic diagram of GEP constructs used to map those ofits fragments that bind to TNFR. (Right) β-Galactosidase assays.

FIG. 10 (Left) Schematic diagram of GEP constructs. (Right)β-Galactosidase assays.

FIG. 11 (Left) Schematic diagram of GEP constructs. (Right)β-Galactosidase assays.

FIG. 12 (Left) Schematic diagram of GEP constructs used to map those ofits fragments that bind to TNFR. (Right) β-Galactosidase assays.

FIG. 13 (Left) Schematic diagram of GEP constructs used to map those ofits fragments that bind to TNFR. (Right) β-Galactosidase assays.

FIG. 14 (Left) Schematic diagram of GEP constructs used to map those ofits fragments that bind to TNFR. (Right) β-Galactosidase assays.

FIG. 15 (Left) Schematic diagram of GEP constructs used to map those ofits fragments that bind to TNFR. (Right) β-Galactosidase assays.

FIG. 16 (A) Atsttrin inhibites the respiratory burst triggered by TNF ina does-dependent manner; (B) Effect of Atsttrin, Remicade and Enbrel onTNF-triggered respiratory burst. Results are means for nmol H2O2produced by 1.5×104 cells/well in triplicate cultures.

FIG. 17 depicts a TUNEL staining assay. Rat chondrosarcoma (RCS) cellsand human C28I2 chondrocytes were serum-starved for 24 h to remove theeffect of exogenous growth factors and cytokines. Thereafter, cells werestimulated with 0.02% BSA (CTR), 50 ng/ml of GEP (GEP), 10 ng/ml ofTNF-α, or GEP plus TNF-α for 36 h. Apoptosis was measured by using anTUNEL assay kit.

FIG. 18 shows that GEP inhibits TNFα-induced metalloproteinase. Humancartilage explants were cultured in the absence or presence of either 5ng/ml of TNFα supplemented with various amount of GEP, as indicated, for1 day in serum-free medium and real-time PCR was performed. The unitsare arbitrary and the leftmost bar in each group indicates a relativelevel of 1.

FIG. 19 A proposed model for explaining the anti-inflammation mechanismsof Atsttrin, Enbrel and Remicade. Atsttrin blocks TNF/TNFR signaling viadirectly binding to TNF receptors, whereas Enbrel and Remicade disturbsignaling via targeting to TNF ligand.

FIG. 20 shows that Atsttrin neutralizes GEP-stimulated cell growth ofcancer cells (MTT assay). RCS chondrosarcoma (left panel) and Saos-2osteosarcoma (right panel) were cultured in the absence (CTR) orpresence of GEP (200 ng/1) with or without various amounts of Atsttrin,as indicated, and cell proliferation was analyzed using an MTT assay.

FIG. 21 depicts IL-6 and IL-13 levels in cell-free exudates of mice froman air-pouch acute inflammation model. Levels of IL-6 and IL-13 aredepicted in controls (CTR) and animals administered Remicade (10 μg/g),GEP (10 μg/g), and Atsttrin (10 μg/g).

FIG. 22 depicts RANKL-induced osteoclastogenesis, as assessed by TRAPstaining. Raw 264.7 macrophages were incubated in the presence of RANKLfor 4 days, alone or with varying amounts of GEP or atsttrin.

FIG. 23 provides the amino acid sequence of human GEP (SEQ ID NO: 1)which is 593 amino acids.

FIG. 24 depicts the sequence of atsttrin peptide (SEQ ID NO: 2), andsequences of various other tested peptides (SEQ ID NOS: 3-7,respectively).

FIGS. 25A and 25B depicts the (A) structure and (B) amino acid sequenceof mouse GEP (SEQ ID NO: 8). In (B), the granulin units GrnA, GrnC, GrnDand GrnE are underlined and indicated at each unit.

FIG. 26 shows that GEP associates with RANK and FAS in addition to TNFR,whereas Atsttrin specifically binds to TNFR (yeast two hybrid assay).Each pair of plasmids, as indicated, was co-transformed into yeaststrain MAV203. Yeast transformants were selected onSD-leu⁻/trp⁻/his⁻/ura⁻/3AT⁺ plates and tested for β-galactosidaseactivity. The lack of interaction between Rb and lamin was used as anegative control (Neg. CTR).

FIG. 27 provides FastStep™ Kinetic Assay for binding of GEP (PGRN) andTNFα to TNFR1 (R1) and TNFR2 (R2).

FIGS. 28A, 28B and 28C provides (A) characterization of recombinantAtsttrin. GST-Atsttrin conjugated to glutathione agarose resin andAtsttrin released by factor Xa were resolved by SDS-PAGE and theproteins were stained with Coomassie Brillian Blue R-250. (B) and (C)provide the FastStep™ Kinetic Assay for binding of Atsttrin and TNFα toTNFR1 (R1) and TNFR2 (R2).

FIG. 29 depicts PathScan® Multiplex Western Blot results against p-AKT,p-ERK and tubulin control with GEP, Atsttrin and GEP+Atsttrin. GEPactivates both the Akt and Erk1/2 pathways, but Atsttrin does not. Inthe combination study, Atsttrin blocked GEP-mediated activation of theoncogenic p-AKT and p-ERK pathways.

FIGS. 30A and 30B depicts Severity (A) (by clinical score) and Incidence(B) of arthritis in CIA mice treated with PBS (n=10), Atsttrin (n=10),or Enbrel (n=10).

FIG. 31 provides photographs of the front (top) and hind (bottom) pawsof normal and CIA mice treated with PBS, Atsttrin, or Enbrel.

FIGS. 32A and 32B depict (A) sections of each ankle of PBS-treated andAtsttrin-treated animals stained with H&E or Safranin-O as indicated. Inthe H&E panel, arrows indicate tissue destruction and cell infiltration,respectively. In the Sarfanin-O panel, arrows indicate loss of matrixstaining. (B) shows MicroCT images of the ankle of PBS-treated andAtsttrin-treated animals.

FIG. 33 provides TRAP staining images in PBS-treated andAtsttrin-treated animals. TRAP+ osteoclasts are depicted in red stainingin the PBS-treated animal but are hardly detectable in theAtsttrin-treated animal. Different magnifications are depicted forvisual emphasis.

FIG. 34 depicts the effects of Atsttrin compared to controls PBS(negative control) and Enbrel (positive control) on proinflammatorycytokines IL-1β and IL-6 and anti-inflammatory cytokines IL-10 andIL-13. The *, **, and *** indicate p<0.05, p<0.01 and p<0.001respectively.

FIG. 35 provides clinical score data in CIA animals treated with eitherPBS, Enbrel or Atsttrin starting 25 days after collagen immunization toinduce arthritis.

FIG. 36 depicts GEP and Atsttrin effects on TNF-induced nitriteproduction. TNFα was added to RAW264.7 cells with concomitant additionof 0.3 nM, 1.5 nM or 7.5 nM GEP, Atsttrin or Enbrel as indicated and μMnitrite was measured.

FIG. 37 evaluates TNF-induced nuclear accumulation of NF-κB byimmunofluorescence of RAW264.7 cells in the presence of TNF-α, TNF-α andGEP, or TNF-α and Atsttrin. Cells were stained for NF-κB p65, versusnuclear DAPI stain, and merged for both stains. NF-κB staining remainscytoplasmic in the presence of GEP or Atsttrin.

FIG. 38 shows fold induction of a TNF-activated NFκB reported gene inthe presence of TNF-α alone, or combined with either GEP or Atsttrin atincreasing concentrations. GEP and Atsttrin inhibit TNF-α mediatedactivation of the NFκB reported gene.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

The terms “Atsttrin”, “Antagonist of TNF/TNFR Signaling via TargetingTNF Receptors”, “atsttrin peptide”, “TNF antagonist peptide” and anyvariants not specifically listed, may be used herein interchangeably,and as used throughout the present application and claims refer topeptides including single or multiple proteins, particularly which arederived from or fragments of GEP and extends to those proteins havingthe amino acid sequence data described herein and presented in FIG. 24and also diagrammed in FIG. 15, and the profile of activities andcapabilities described and set forth herein and provided in the Claims.Active GEP peptides having activity as antagonist of TNF/TNFR signalingand capable of binding one or more TNF receptors, such as TNFα, areincluded and provided herein. The full length sequence of human GEP isprovided in FIG. 23. The full length sequence of mouse GEP is providedin FIG. 25. Thus, TNF antagonist peptides derived from GEP sequences(s)or comprising GEP sequence(s) and having TNF and/or TNF-familyantagonist activity are encompassed herein. These atsttrin peptidesinclude and encompass fragments, variants, and derivatives of thepeptides. Accordingly, proteins displaying substantially equivalentactivity, and which are modifications thereof, are likewisecontemplated. These modifications may be deliberate, for example, suchas modifications obtained through site-directed mutagenesis, or may beaccidental, such as those obtained through mutations in hosts that areproducers of the complex or its named subunits. Corresponding mouse orother species or ortholog GEP sequences to the human atsttrin and activeGEP peptide sequences are further contemplated. Also, the terms“Atsttrin”, “Antagonist of TNF/TNFR Signaling via Targeting TNFReceptors”, “atsttrin peptide”, “TNF antagonist peptide” are intended toinclude within their scope proteins specifically recited herein as wellas all substantially homologous analogs and allelic variations.

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfuctional property of immunoglobulin-binding is retained by thepolypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969),abbreviations for amino acid residues are shown in the following Tableof Correspondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid W Trp tryptophan R Arg arginine D Asp aspartic acid NAsn asparagine C Cys cysteine

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues. The above Table ispresented to correlate the three-letter and one-letter notations whichmay appear alternately herein.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. It should be appreciated thatalso within the scope of the present invention are DNA sequencesencoding which code for a having the same amino acid sequence as SEQ IDNO:, but which are degenerate to SEQ ID NO:. By “degenerate to” is meantthat a different three-letter codon is used to specify a particularamino acid. It is well known in the art that certain codons can be usedinterchangeably to code for each specific amino acid. For example, andnot by limitation, Leucine (Leu or L) may be encoded by any of UUA orUUG or CUU or CUC or CUA or CUG, and Serine (Ser or S) may be encoded byany of UCU or UCC or UCA or UCG or AGU or AGC. It should be understoodthat these exemplary codons specified are for RNA sequences. Thecorresponding codons for DNA have a T substituted for U.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20^(N)C below the predicted or determined T_(m) with washes of higherstringency, if desired.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “l” means liter.

Mutations can be made in GEP, GEP peptides, and/or atsttrin such anamino acid is substituted or modified or such that a particular codon ischanged to a codon which codes for a different amino acid. Such amutation is generally made by making the fewest nucleotide changespossible. A substitution mutation of this sort can be made to change anamino acid in the resulting protein in a non-conservative manner (i.e.,by changing the codon from an amino acid belonging to a grouping ofamino acids having a particular size or characteristic to an amino acidbelonging to another grouping) or in a conservative manner (i.e., bychanging the codon from an amino acid belonging to a grouping of aminoacids having a particular size or characteristic to an amino acidbelonging to the same grouping). Such a conservative change generallyleads to less change in the structure and function of the resultingprotein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein or peptide. The presentinvention should be considered to include sequences containingconservative and/or non-conservative changes which do not significantlyalter the activity or binding characteristics of the resulting proteinor peptide.

The following is one example of various groupings of amino acids:

-   Amino acids with nonpolar R groups: Alanine, Valine, Leucine,    Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine-   Amino acids with uncharged polar R groups: Glycine, Serine,    Threonine, Cysteine, Tyrosine, Asparagine, Glutamine-   Amino acids with charged polar R groups (negatively charged at Ph    6.0): Aspartic acid, Glutamic acid-   Basic amino acids (positively charged at pH 6.0): Lysine, Arginine.    Histidine (at pH 6.0)-   Another grouping may be those amino acids with phenyl groups:    Phenylalanine, Tryptophan, Tyrosine.

Another grouping may be according to molecular weight (i.e., size of Rgroups):

Glycine 75 Alanine 89 Serine 105 Proline 115 Valine 117 Threonine 119Cysteine 121 Leucine 131 Isoleucine 131 Asparagine 132 Aspartic acid 133Glutamine 146 Lysine 146 Glutamic acid 147 Methionine 149 Histidine (atpH 6.0) 155 Phenylalanine 165 Arginine 174 Tyrosine 181 Tryptophan 204

-   -   Particularly preferred substitutions are:    -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free NH₂ can be maintained.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site for disulfide bridges with another Cys. AHis may be introduced as a particularly “catalytic” site (i.e., His canact as an acid or base and is the most common amino acid in biochemicalcatalysis). Pro may be introduced because of its particularly planarstructure, which induces turns in the protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

An “antibody” is any immunoglobulin, including antibodies and fragmentsthereof, that binds a specific epitope. The term encompasses polyclonal,monoclonal, and chimeric antibodies, the last mentioned described infurther detail in U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′)₂ and F(v), which portionsare preferred for use in the therapeutic methods described herein.

Fab and F(ab′)₂ portions of antibody molecules are prepared by theproteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′)₂ portions followed by reduction of the disulfide bonds linkingthe two heavy chain portions as with mercaptoethanol, and followed byalkylation of the resulting protein mercaptan with a reagent such asiodoacetamide. An antibody containing intact antibody molecules ispreferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The term “therapeutically effective amount” means that amount of a drug,compound, peptide, or pharmaceutical agent that will elicit thebiological or medical response of a subject that is being sought by amedical doctor or other clinician. The phrase “therapeutically effectiveamount” is used herein to include an amount sufficient to prevent, andpreferably reduce by at least about 30 percent, more preferably by atleast 50 percent, most preferably by at least 90 percent, a clinicallysignificant change in the S phase activity of a target cell or cellularmass, or other feature of pathology such as for example, elevated bloodpressure, fever or white cell count as may attend its presence andactivity.

The term “preventing” or “prevention” refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term “prophylaxis” is related to and encompassed in the term“prevention”, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

The term “solvate” means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. “Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term “subject” includes humans and other mammals.

The term “treating” or “treatment” of any disease or disorder refers, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment‘treating’ or ‘treatment’ refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, ‘treating’ or ‘treatment’ refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, ‘treating’ or ‘treatment’relates to slowing the progression of the disease.

The term “disease characterized by inflammation”, “inflammatory disease”refers to a disease which involves, results at least in part from, orincludes inflammation. The term includes, but is not limited to,exemplary diseases selected from rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, juvenile idiopathic arthritis, psoriasis,inflammatory bowel diseases, Chrohn's disease, ulcerative colitis,uveitis, inflammatory lung diseases, chronic obstructive pulmonarydisease.

The term “inflammatory mediators” refers to mediators which enhance,initiate or facilitate an inflammatory reaction or an inflammatoryresponse, and may be selected from the following: Cytokines (e.g.TNFalpha, IL3, IL4, IL5, IL13, GM-CSF), chemokines (e.g. MDC, CCL19,CCL20, CCL21, MIP-1alpha), Prostaglandins (e.g. PGD2), Leukotrienes(e.g. LTB4, LTC4, LTD4), metalloproteases, chymase, tryptase, growthfactors (e.g. VEGF).

Despite the prevalence of arthritic diseases, their precise etiology,pathogenesis, and progression remain beyond our understanding. Evidenceis accumulating that demonstrates the significance of inflammatorycytokines and growth factors in the pathological processes of arthritis.The isolation of the growth factors that regulate chondrocytes andarthritis, and inhibitors that antagonize the actions of cytokines, aretherefore of great importance from both a pathophysiological and atherapeutic standpoint. Granulin/epithelin precursor (GEP) has beenpreviously recognized as a novel chondrogenic growth factor that playsan essential role in cartilage formation, including as described in WO2008/094687 A2 and by Liu and colleagues (Xu, K et al (2007) J Biol Chem282(15):11347-11355).

The present invention demonstrates that the growth factor GEP directlyassociates with TNF receptors and acts as a naturally-occurringantagonist of TNFα, the central inflammatory cytokine in arthritis.Thus, the purpose of this invention is to extend the understanding ofthe molecular mechanisms by which growth factors and cytokines controlcartilage development and arthritis, and to provide GEP, particularlyits derived and active peptide(s), particularly atsttrin, and/orderivatives or variants thereof as novel anti-TNF/TNFR modulators.Atsttrin is demonstrated herein to bind and antagonize TNF, and alterTNF/TNFR signaling. GEP is demonstrated herein to bind RANK a TNF familymember. Thus, GEP, GEP peptides, and/or atsttrin are applicable anduseful in therapeutic interventions for various kinds of TNF-relateddiseases, including inflammatory conditions such as arthritis, bonediseases, and cancer conditions, such as osteoarthritis, osteoporosis,and osteosarcoma.

In vivo animal models of TNF/TNFR family mediated diseases or conditionsmay be utilized by the skilled artisan to further or additionallyevaluate, assess, screen and/or verify the GEP, GEP peptides and/oratsttrin of the invention or agents or compounds identified in or inaccordance with the present invention, including further assessingTNF/TNFR modulation in vivo. Animal models are readily available todemonstrate the applicability of recombinant GEP and GEP-derivedpeptide(s), atsttrin in mediating, alleviating or controlling thedevelopment and progression of TNF/TNFR mediated diseases or conditions,inflammatory conditions, immune diseases or conditions (includingallergies an auto-immune diseases), bone diseases or conditions, orother possible targeted conditions. Animal models or studies includethose described and detailed herein and in the examples. TNFα transgenicmice develop arthritis and provide a useful tool for evaluating theefficacy of novel therapeutic strategies for rheumatoid arthritis.Animal models include, but are not limited to, ulcerative colitismodels, multiple sclerosis models (including EAE, lysolecithin-induced),arthritis models, allergic asthma models, airway inflammation models,psoriasis models, and acute inflammation models. The EAE animal model ofmultiple sclerosis provides an acute or chronic-relapsing, acquired,inflammatory and demyelinating autoimmune disease. Allergy models may beutilized as models of immunological injury and conditions.Osteoarthritis models include for example experimental osteoarthritisinduced in rabbits after sectioning of the knee anterior cruciateligament and in rats after tear of the medial collateral ligament.Appropriate bone disease, bone injury, and/or osteoporosis models arealso known and available to one of skill in the art.

The invention includes use and applications of GEP, GEP peptides,atsttrin, and/or derivatives thereof for prevention, treatment oralleviation of rheumatoid arthritis and osteoarthritis. The inventionincludes use and applications of GEP, GEP peptides, atsttrin, and/orderivatives thereof for prevention, treatment or alleviation ofTNF-related diseases, including inflammatory conditions, immuneconditions including auto-immune diseases, bone diseases and cancer.TNF-related diseases include rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, psoriasis, inflammatory bowel diseases, Chrohn'sdisease, ulcerative colitis, uveitis, inflammatory lung diseases,chronic obstructive pulmonary disease, multiple sclerosis, osteoporosis,osteosarcoma. The invention includes use and applications of GEP, GEPpeptides, atsttrin, and/or derivatives thereof for prevention, treatmentor alleviation of and/or for specific therapeutic intervention ofinflammatory disorders by delivering precisely the requiredanti-TNF/TNFR effect. The invention includes use and applications ofGEP, GEP peptides, atstrrin, and/or derivatives thereof for facilitatingor mediating tissue repair. The invention includes use and applicationsof GEP, GEP peptides, atsttrin, and/or derivatives thereof forprevention, treatment or alleviation of immunological injury andconditions, including allergies and auto-immune diseases, such as lupusand multiple sclerosis. The invention includes use and applications ofGEP, GEP peptides, atsttrin, and/or derivatives thereof for prevention,treatment or alleviation of cancer and tumor or cancer cell growth,including in GEP and/or TNF/TNFR mediated cancers or other suchhyperproliferative disorders.

The possibilities both diagnostic and therapeutic that are raised by theexistence of TNF antagonist peptides, including GEP, GEP peptides and/oratsttrin as described herein, derive from the fact that the peptidesparticipate in direct and causal protein-protein interaction with TNFand serve to block, inhibit, antagonize, interfere with TNF/TNFRactivity and/or signaling. Thus, the present invention contemplatespharmaceutical intervention in the cascade of reactions in whichTNF/TNFR and/or TNF family/TNF family R is implicated, to modulate theactivity, signal(s), and/or conditions initiated, facilitated ormediated thereby, including but not limited to inflammatory diseases andconditions.

The GEP, GEP peptides and/or atsttrin as described herein or otherligands or agents exhibiting either mimicry therewith or cognate TNFantagonism, may be prepared in pharmaceutical compositions, with asuitable carrier and at a strength effective for administration byvarious means to a patient experiencing an adverse medical conditionassociated with TNF/TNFR signaling, such as an inflammatory condition ordisease. A variety of administrative techniques may be utilized, amongthem parenteral techniques such as subcutaneous, intravenous andintraperitoneal injections, catheterizations and the like. Averagequantities of the GEP, GEP peptides and/or atsttrin as described hereinor their subunits may vary and in particular should be based upon therecommendations and prescription of a qualified physician orveterinarian.

Also, antibodies including both polyclonal and monoclonal antibodies,and drugs that modulate the production or activity of the GEP, GEPpeptides and/or atsttrin as described herein and/or their subunits maypossess certain diagnostic applications and may for example, be utilizedfor the purpose of detecting and/or measuring conditions such asTNF-mediated diseases, inflammatory conditions, infections, cancer, orthe like. For example, the GEP peptides and/or atsttrin may be used toproduce both polyclonal and monoclonal antibodies to themselves in avariety of cellular media, by known techniques such as the hybridomatechnique utilizing, for example, fused mouse spleen lymphocytes andmyeloma cells. Likewise, small molecules that mimic or antagonize theactivity(ies) of the GEP, GEP peptides and/or atsttrin as describedherein of the invention may be discovered or synthesized, and may beused in diagnostic and/or therapeutic protocols.

The general methodology for making monoclonal antibodies by hybridomasis well known. Immortal, antibody-producing cell lines can also becreated by techniques other than fusion, such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980);Hammerling et al., “Monoclonal Antibodies And T-cell Hybridomas” (1981);Kennett et al., “Monoclonal Antibodies” (1980); see also U.S. Pat. Nos.4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917;4,472,500; 4,491,632; 4,493,890.

Preferably, the anti-GEP, GEP peptides and/or atsttrin antibody used inthe diagnostic methods of this invention is an affinity purifiedpolyclonal antibody. More preferably, the antibody is a monoclonalantibody (mAb). In addition, it is preferable for the anti-GEP, GEPpeptides and/or atsttrin antibody molecules used herein be in the formof Fab, Fab′, F(ab)₂ or F(v) portions of whole antibody molecules.

The present invention further contemplates therapeutic compositionsuseful in practicing the therapeutic methods of this invention. Atherapeutic composition includes a biologically compatible composition.A subject therapeutic composition includes, in admixture, apharmaceutically acceptable excipient (carrier) and one or more of aGEP, GEP peptide(s) and/or atsttrin polypeptide analog thereof orfragment thereof, as described herein as an active ingredient. In apreferred embodiment, the composition comprises the present GEP, GEPpeptide(s) and/or atsttrin, and may comprise composition comprising oneor more GEP granulin and one or more linker unit, or any one or more ofthe GEP peptide(s) or atsttrin, including as set out in the figuresherein, including FIGS. 23 and 24 and as provided in SEQ ID NOS: 1-8.

The peptides and compositions of the invention include those GEPpeptides, including atsttrin, which are based on the human GEP sequence,including as set out in FIGS. 23 and 34, as well as variants thereofhaving one or more or a few or many substitutions, wherein the bindingand activity profiles of the variant(s) are retained when compared tothe atsttrin GEP peptide. In as much as GEP peptides from variousanimals or mammals, including humans, are known, these sequences providealternative amino acid sequences and variants of potential use in thecompositions and methods of the invention, including by substitution ofsome of the atsttrin human peptide amino acids. Mouse GEP sequence isprovided herein in FIG. 25. GEP sequences for various animals arepublicly known and disclosed and would be available for evaluation andassessment in the methods and compositions of the invention, and theircorresponding and correlating amino acids suitable for evaluation anduse as variants of the GEP peptides herein. GEP sequences are availableand herein incorporated by reference as follows: rat (Genbank accessionAAA16903.1, CAA44198.1), mouse (Genbank accession P28798.2, BAE35389.1,NP_(—)032201.2), Sumatran orangutan (Genbank accessionNP_(—)001126689.1), crab-eating macague (Genbank accession BAE01796.1),horse (Genbank accession XP_(—)001489791.1), cattle (Genbank accessionNP_(—)001070482.1), rabbit (Genbank accession XP_(—)002719228.1), pig(Genbank accession NP_(—)001038043.1), chimpanzee (Genbank accessionXP_(—)511549.2) and opossum (Genbank accession XP_(—)001374870.1).

A peptide, analog or active fragment can be formulated into thetherapeutic composition as neutralized pharmaceutically acceptable saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from thefree carboxyl groups can also be derived from inorganic bases such as,for example, sodium, potassium, ammonium, calcium, or ferric hydroxides,and such organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

The therapeutic peptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of TNF/TNFR activity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations of ten nanomolar to ten micromolar in the blood arecontemplated.

A particular biologically compatible composition is an aqueous solutionthat is buffered using, e.g., Tris, phosphate, or HEPES buffer,containing salt ions. Usually the concentration of salt ions will besimilar to physiological levels. Biologically compatible solutions mayinclude stabilizing agents and preservatives. In a more preferredembodiment, the biocompatible composition is a pharmaceuticallyacceptable composition. Such compositions can be formulated foradministration by topical, oral, parenteral, intranasal, subcutaneous,and intraocular, routes. Parenteral administration is meant to includeintravenous injection, intramuscular injection, intraarterial injectionor infusion techniques. The composition may be administered parenterallyin dosage unit formulations containing standard, well-known non-toxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

A particular embodiment of the present composition invention is apharmaceutical composition comprising a therapeutically effective amountof GEP, GEP peptide(s) and/or atsttrin as described hereinabove, inadmixture with a pharmaceutically acceptable carrier. Another particularembodiment is a pharmaceutical composition for the treatment orprevention of a disease characterized by TNF/TNFR activity includinginfections, allograft reactions, inflammation, allergic and autoimmunediseases, and cancer, or a susceptibility to said disease, comprising aneffective amount of the GEP, GEP peptide(s) and/or atsttrin, itspharmaceutically acceptable salts, hydrates, solvates, or prodrugsthereof in admixture with a pharmaceutically acceptable carrier. Afurther particular embodiment is a pharmaceutical composition for thetreatment or prevention of a disease involving inflammation, or asusceptibility to the condition, comprising an effective amount of theGEP, GEP peptide(s) and/or atsttrin, its pharmaceutically acceptablesalts, hydrates, solvates, or prodrugs thereof in admixture with apharmaceutically acceptable carrier.

The compositions of the invention may include GEP, GEP peptides,atsttrin, and/or derivatives thereof in combination with one or moreagents suitable for the alleviation, prevention or treatment ofinflammation, immunological conditions, hyperproliferative conditions,and/or cancer. The compositions of the invention may include GEP, GEPpeptides, atsttrin, and/or derivatives thereof in combination with oneor more of an anti-inflammatory agent, an anti-cancer agent, or animmunodulatory agent. More generally these anti-cancer agents may betyrosine kinase inhibitors or phosphorylation cascade inhibitors,post-translational modulators, cell growth or division inhibitors (e.g.anti-mitotics), inhibitors or signal transduction inhibitors. Othertreatments or therapeutics may include the administration of suitabledoses of pain relief drugs such as non-steroidal anti-inflammatory drugs(e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such asmorphine, or anti-emetics. In addition, the composition may incorporateor be administered with immune modulators, such as interleukins, tumornecrosis factor (TNF) or other growth factors, colony stimulatingfactors, cytokines or hormones.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Pharmaceutical compositions for oral usecan be prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Preferred sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium; calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The agents or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Particularly, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treatment with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In theparticular embodiment, the matrix is biodegradable over a time period ofless than a year, more particularly less than six months, mostparticularly over two to ten weeks. The polymer composition, as well asmethod of manufacture, can be used to determine the rate of degradation.For example, mixing increasing amounts of polylactic acid withpolyglycolic acid decreases the degradation time. Meshes of polyglycolicacid that can be used can be obtained commercially, for instance, fromsurgical supply companies (e.g., Ethicon, N.J.). In general, thesepolymers are at least partially soluble in aqueous solutions, such aswater, buffered salt solutions, or aqueous alcohol solutions that havecharged side groups, or a monovalent ionic salt thereof.

The composition medium can also be a hydrogel, which is prepared fromany biocompatible or non-cytotoxic homo- or hetero-polymer, such as ahydrophilic polyacrylic acid polymer that can act as a drug absorbingsponge. Certain of them, such as, in particular, those obtained fromethylene and/or propylene oxide are commercially available. A hydrogelcan be deposited directly onto the surface of the tissue to be treated,for example during surgical intervention.

The active expression-inhibiting agents may also be entrapped inmicrocapsules prepared, for example, by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofprotein, polynucleotide, peptide, or its antibodies, agonists orantagonists, which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED₅₀ (the dose therapeutically effective in 50% of the population)and LD₅₀ (the dose lethal to 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions thatexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used in formulating a rangeof dosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to target tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to target cells by other methods known inthe art. Localized administration to the desired tissues may be done bydirect injection, transdermal absorption, catheter, infusion pump orstent. The DNA, DNA/vehicle complexes, or the recombinant virusparticles are locally administered to the site of treatment. Alternativeroutes of delivery include, but are not limited to, intravenousinjection, intramuscular injection, subcutaneous injection, aerosolinhalation, oral (tablet or pill form), topical, systemic, ocular,intraperitoneal and/or intrathecal delivery.

Alternatively, or additionally, a polynucleotide encoding the GEP, GEPpeptide(s) and/or atsttrin may be particularly included within a vector.The polynucleic acid is operably linked to signals enabling expressionof the nucleic acid sequence and is introduced into a cell utilizing,preferably, recombinant vector constructs, which will express theantisense nucleic acid once the vector is introduced into the cell. Avariety of viral-based systems are available, including adenoviral,retroviral, adeno-associated viral, lentiviral, herpes simplex viral ora sendaiviral vector systems, and all may be used to introduce andexpress polynucleotide sequence for the expression-inhibiting agents orthe polynucleotide expressing the TARGET polypeptide in the targetcells.

Particularly, the viral vectors used in the methods of the presentinvention are replication defective. Such replication defective vectorswill usually pack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or addition ofone or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid apreferred vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the target cell population that an adenoviral capsidof the invention can enter in a preferred embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51.Techniques or construction and expression of these chimeric vectors aredisclosed in US 2003/0180258 and US 2004/0071660, hereby incorporated byreference.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto target the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“murine Moloney leukemia virus”) MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention.

In other embodiments of the present invention, adeno-associated viruses(“AAV”) are utilized. The AAV viruses are DNA viruses of relativelysmall size that integrate, in a stable and site-specific manner, intothe genome of the infected cells. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP_(r), P₁, and trp promoters. Among the eukaryotic (including viral)promoters useful for practice of this invention are ubiquitous promoters(e.g. HPRT, vimentin, actin, tubulin), therapeutic gene promoters (e.g.MDR type, CFTR, factor VIII), tissue-specific promoters, includinganimal transcriptional control regions, which exhibit tissue specificityand have been utilized in transgenic animals, e.g. immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl, et al.(1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8;Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), and mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95).

Other promoters which may be used in the practice of the inventioninclude promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40, E1a, and MLP promoters. Further promoters whichmay be of use in the practice of the invention include promoters whichare active and/or expressed in dendritic cells.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient. For example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Feigner, et. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Feigner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described inInternational Patent Publications WO 95/18863 and WO 96/17823, and inU.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenousgenes into the specific organs in vivo has certain practical advantagesand directing transfection to particular cell types would beparticularly advantageous in a tissue with cellular heterogeneity, forexample, pancreas, liver, kidney, and the brain. Lipids may bechemically coupled to other molecules for the purpose of targeting.Targeted peptides, e.g., hormones or neurotransmitters, and proteins forexample, antibodies, or non-peptide molecules could be coupled toliposomes chemically. Other molecules are also useful for facilitatingtransfection of a nucleic acid in vivo, for example, a cationicoligopeptide (e.g., International Patent Publication WO 95/21931),peptides derived from DNA binding proteins (e.g., International PatentPublication WO 96/25508), or a cationic polymer (e.g., InternationalPatent Publication WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). NakedDNA vectors for therapeutic purposes can be introduced into the desiredhost cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, use of a gene gun, or use of aDNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem.267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al.Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990;Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30).Receptor-mediated DNA delivery approaches can also be used (Curiel, etal. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem.262:4429-32).

In addition, the present invention envisions preparing GEP peptidesand/or atsttrin peptides that have distinct or different structural orsequence properties, and the use of peptidomimetics, and peptidomimeticbonds, such as ester bonds, to prepare additional peptides or agentswith the properties of the GEP peptides and/or atsttrin, i.e. capable ofinhibiting or antagonizing TNF and TNF/TNFR. In another embodiment, apeptide may be generated that incorporates a reduced peptide bond, i.e.,R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues or sequences. Areduced peptide bond may be introduced as a dipeptide subunit. Such amolecule would be resistant to peptide bond hydrolysis, e.g., proteaseactivity. Such peptides would provide antagonists with unique functionand activity, such as extended half-lives in vivo due to resistance tometabolic breakdown, or protease activity. Furthermore, it is well knownthat in certain systems constrained peptides show enhanced functionalactivity (Hruby, 1982, Life Sciences 31:189-199; Hruby et al., 1990,Biochem J. 268:249-262.

A constrained, cyclic or rigidized peptide may be preparedsynthetically, provided that in at least two positions in the sequenceof the peptide an amino acid or amino acid analog is inserted thatprovides a chemical functional group capable of cross-linking toconstrain, cyclise or rigidize the peptide after treatment to form thecross-link. Cyclization will be favored when a turn-inducing amino acidis incorporated. Examples of amino acids capable of cross-linking apeptide are cysteine to form disulfide, aspartic acid to form a lactoneor a lactase, and a chelator such as γ-carboxyl-glutamic acid (Gla)(Bachem) to chelate a transition metal and form a cross-link. Protectedγ-carboxyl glutamic acid may be prepared by modifying the synthesisdescribed by Zee-Cheng and Olson (1980, Biophys. Biochem. Res. Commun.94:1128-1132). A peptide in which the peptide sequence comprises atleast two amino acids capable of cross-linking may be treated, e.g., byoxidation of cysteine residues to form a disulfide or addition of ametal ion to form a chelate, so as to cross-link the peptide and form aconstrained, cyclic or rigidized peptide.

The present invention contemplates strategies to systematically preparecross-links. For example, if four cysteine residues are incorporated inthe peptide sequence, different protecting groups may be used (Hiskey,1981, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross andMeienhofer, eds., Academic Press: New York, pp. 137-167; Ponsanti etal., 1990, Tetrahedron 46:8255-8266). The first pair of cysteine may bedeprotected and oxidized, then the second set may be deprotected andoxidized. In this way a defined set of disulfide cross-links may beformed. Alternatively, a pair of cysteine and a pair of collating aminoacid analogs may be incorporated so that the cross-links are of adifferent chemical nature.

The following non-classical amino acids may be incorporated in thepeptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991,J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, TetrahedronLett.); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989,Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.,1989, J. Takeda Res. Labs. 43:53-76); β-carboline (D and L) (Kazmierski,1988, Ph.D. Thesis, University of Arizona); HIC (histidine isoquinolinecarboxylic acid) (Zechel et al., 1991, Int. J. Pep. Protein Res. 43);and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2_propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al., 1985, J. Org. Chem. 50:5834-5838);β-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett.29:5081-5082); β-turn inducing analogs (Kemp et al., 1988, TetrahedronLett. 29:5057-5060); ∝_helix inducing analogs (Kemp et al., 1988,Tetrahedron Lett. 29:4935-4938); γ-turn inducing analogs (Kemp et al.,1989, J. Org. Chem. 54:109:115); and analogs provided by the followingreferences: Nagai and Sato, 1985, Tetrahedron Lett. 26:647_(—)650;DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p. 1687; also a Gly-Alaturn analog (Kahn et al., 1989, Tetrahedron Lett. 30:2317); amide bondisostere (Jones et al., 1988, Tetrahedron Lett. 29:3853-3856); tretrazol(Zabrocki et al., 1988, J. Am. Chem. Soc. 110:5875-5880); DTC (Samanenet al., 1990, Int. J. Protein Pep. Res. 35:501:509); and analogs taughtin Olson et al., 1990, J. Am. Chem. Sci. 112:323-333 and Garvey et al.,1990, J. Org. Chem. 56:436. Conformationally restricted mimetics of betaturns and beta bulges, and peptides containing them, are described inU.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

The present invention further provides for modification orderivatization of the polypeptide or peptide of the invention. Thesemodifications may serve to alter or increase the stability, activity,half-life of the polypeptide or peptide of the invention. Modificationsof peptides are well known to one of ordinary skill, and includephosphorylation, carboxymethylation, and acylation. Modifications may beeffected by chemical or enzymatic means. In another aspect, glycosylatedor fatty acylated peptide derivatives may be prepared. Preparation ofglycosylated or fatty acylated peptides is well known in the art. Fattyacyl peptide derivatives may also be prepared. For example, and not byway of limitation, a free amino group (N-terminal or lysyl) may beacylated, e.g., myristoylated. In another embodiment an amino acidcomprising an aliphatic side chain of the structure —(CH₂)_(n)CH₃ may beincorporated in the peptide. This and other peptide-fatty acidconjugates suitable for use in the present invention are disclosed inU.K. Patent GB-8809162.4, International Patent ApplicationPCT/AU89/00166, and reference 5, supra. Addition of carbohydratemoieties and the preparation and use of glycosylated analogs of thepeptides of the invention is also contemplated, including for improvedbiological and physical properties such as proteolytic stability and invivo activity.

Chemical Moieties For Derivatization. Derivatives of the peptides(including variants, analogs and active fragments thereof) of thepresent invention are further provided. Such derivatives encompass andinclude derivatives to enhance activity, solubility, effectivetherapeutic concentration, and transport across the blood brain barrier.Further encompassed derivatives include the attachment of moieties ormolecules which are known to interact with TNF/TNFR, to target TNF/TNFRor expressing cells, or to have anti-inflammatory activity. The chemicalmoieties may be N-terminally or C-terminally attached to the peptides ofthe present invention. Chemical moieties suitable for derivatization maybe, for instance, selected from among water soluble polymers. Thepolymer selected can be water soluble so that the component to which itis attached does not precipitate in an aqueous environment, such as aphysiological environment. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable. The polymer may be branched or unbranched. One skilled inthe art will be able to select the desired polymer based on suchconsiderations as whether the polymer/component conjugate will be usedtherapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. For the presentcomponent or components, these may be ascertained using the assaysprovided herein.

The water soluble polymer may be selected from the group consisting of,for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayhave advantages in manufacturing due to its stability in water.

The polymer may be of any suitable molecular weight, and may be branchedor unbranched. For polyethylene glycol, the preferred molecular weightis between about 2 kDa and about 100 kDa (the term “about” indicatingthat in preparations of polyethylene glycol, some molecules will weighmore, some less, than the stated molecular weight) for ease in handlingand manufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivative, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to component or componentsmolecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted component or componentsand polymer) will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the component or components with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384 herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., 1992, Exp. Hematol. 20:1028-1035 (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group include lysine residues and theterminal amino acid residues; those having a free carboxyl group includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydrl groups may also be used as a reactive group forattaching the polyethylene glycol molecule(s). Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

More particularly the present invention provides derivatives which arefusion proteins comprising the peptides of the present invention orfragments thereof. Thus peptides of the present invention and fragmentsthereof can be “modified” i.e., placed in a fusion of chimeric peptideor protein, or labeled, e.g., to have an N-terminal FLAG-tag. In aparticular embodiment a peptide can be modified by linkage or attachmentto a marker protein such as green fluorescent protein as described inU.S. Pat. No. 5,625,048 filed Apr. 29, 1997 and WO 97/26333, publishedJul. 24, 1999 (each of which are hereby incorporated by reference hereinin their entireties). In one such embodiment, a chimeric peptide can beprepared, e.g., a glutathione-S-transferase (GST) fusion protein, amaltose-binding (MPB) protein fusion protein, or a poly-histidine-taggedfusion protein, for expression in a eukaryotic cell. Expression of thepeptide of the present invention as a fusion protein can facilitatestable expression, or allow for purification based on the properties ofthe fusion partner. For example, GST binds glutathione conjugated to asolid support matrix, MBP binds to a maltose matrix, and poly-histidinechelates to a Ni-chelation support matrix. The fusion protein can beeluted from the specific matrix with appropriate buffers, or by treatingwith a protease specific for a cleavage site usually engineered betweenthe peptide and the fusion partner (e.g., GST, MBP, or poly-His).Alternatively the chimeric peptide may contain the green fluorescentprotein, and be used to determine the intracellular localization of thepeptide in the cell.

The invention also includes derivatives wherein at least one of theattached chemical moieties is a molecule having multiple sites forpeptide attachment and capable of binding at least two of said peptidessimultaneously to generate a multimeric peptide structure. Thisderivative has the effect of increasing the available localconcentration of the carbohydrate epitope mimic peptide(s) of thepresent invention. Alternatively, or in addition, such moieties canfunction in providing a stable scaffold to retain the peptide in placefor activity, thereby reducing or preventing diffusion or degradation.More particularly, such molecule is selected from the group of BSA,ovalbumin, human serum allbumin, polyacrylamide, beads and syntheticfibers (biodegradable and non-biodegradable).

The carbohydrate epitope mimic peptide of the present invention may beprepared and utilized as monomers, dimers, multimers, heterodimers,heteromultimers, etc. Presentation or administration of the peptide inmultimeric form may result in enhanced activity or otherwise increasedmodulation of the activity mediated by the peptide(s), including TNFantagonistic activity and/or inhibiting TNF/TNFR signaling and activity.The peptide monomer could be produced in a variety of ways. The peptideof the present invention can be synthesized using a protein synthesizerand utilizing methods well known in the art and as describedhereinabove, incorporating amino acid modifications, analogs, etc. ashereinabove described. In addition, the DNA sequence of the peptide canbe inserted into an expression vector such as pSE (Invitrogen) or pcDNA3(Invitrogen) for production in bacterial or mammalian cell expressionsystems. Insect or yeast expression systems could also be used.Purification of the peptide could be facilitated by the addition of atag sequence such as the 6-Histidine tag which binds to Nickel-NTAresins. These tag sequences are often easily removed by the addition ofa protease specific sequence following the tag. Dimers and multimers ofthe peptide can be produced using a variety of methods in the art. TheDNA sequence of a dimer or multimer could also be inserted into anexpression system such as bacteria or mammalian cell systems. This couldproduce molecules such as Met-FLHTRLFV)_(x) where x=2, 3, 4, . . . etc.It may be necessary to include a short flexible spacer(Gly-Gly-Gly-Gly-Ser)₃ between the peptide or peptidomimetic to increaseits effectiveness. Dimers and multimers can also be generated usingcrosslinking reagents such as Disuccinimidyl suberate (DSS) orDithoiobis (succinimidyl propionate) (DSP). These reagents are reactivewith amino groups and could crosslink the peptide through free aminegroups at the arginine residues and the free amine group at theN-terminus. Dimers and multimers can also be formed using affinityinteractions between biotin and avidin, Jun and Fos, and the Fc regionof antibodies. The purified peptide can be biotinylated and mixed withfactors that are known to form strong protein-protein interactions. Thepeptide or peptidomimetic could be linked to the regions in Jun and Fosresponsible for dimer formation using crosslinkers such as thosementioned above or using molecular techniques to create apeptide-Jun/Fos molecule. When the Jun and Fos peptide hybrids aremixed, dimer formation would result. In addition, production of apeptide-Fc hybrid could also be produced. When expressed in mammaliancells, covalent disulfide bonds form through cysteines in the Fc regionand dimer formation would result. Heterodimers and heteromultimers ofthe peptide could also be produced. This would generate possiblemultifunctional molecules where parts of the whole molecule areresponsible for producing a multitude of effects, such as anti-TNFand/or anti-inflammatory and/or cell growth modulating effects. The sametechnologies as those listed above could be used to generate thesemultifunctional molecules. Molecular techniques could be used to insertthe carbohydrate epitope mimic peptide into a protein at the DNA level.This insertion could take place at the N- or C-terminus, or in themiddle of the protein molecule. Heterodimers could be formed usingpeptide/Fc or peptide/June or Fos hybrid molecules. When mixed withother Fc or Jun/Fos containing hybrids dimer formation would resultproducing heterodimers. Crosslinking reagents could also be used to linkthe peptide to heterodimers. Lastly, biotinylation of the peptide alongwith biotinylation of other molecules could be used to create multimers.Mixing of these components with avidin could create largemultifunctional complexes, where each of the four biotin binding sitesof the avidin molecule is occupied by a different biotinylated molecule.

In one aspect the present invention provides a method of preventingand/or treating a disease characterized by, mediated by or facilitatedby TNF/TNFR activity and/or signaling and/or a diseases characterized byinflammation, immune injury, and cancer, said method comprisingadministering to a subject a therapeutically effective amount of a TNFantagonist peptide as disclosed herein. In a particular embodiment, thepeptide is selected from GEP, GEP peptide(s) and/or atsttrin. In aparticular embodiment, the disease is selected from rheumatoidarthritis, osteoarthritis, ankylosing spondylitis, psoriasis,inflammatory bowel diseases, Chrohn's disease, ulcerative colitis,uveitis, inflammatory lung diseases, and chronic obstructive pulmonarydisease. In an embodiment, the disease may be an immunological disorderor condition, including allergies and auto-immune diseases, such aslupus and multiple sclerosis. In an aspect, the disease may be cancer,including a GEP-mediated cancer or TNF/TNFR mediated cancer.

The invention also relates to the use of a peptide as described aboveand herein for the preparation of a medicament for treating orpreventing a disease characterized by, mediated by or facilitated byTNF/TNFR activity and/or signaling and/or diseases characterized byinflammation, and cancer. In a particular embodiment, the disease ischaracterised by inflammation. In a particular embodiment of the presentinvention the disease is selected from rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, psoriasis, inflammatory boweldiseases, Chrohn's disease, ulcerative colitis, uveitis, inflammatorylung diseases, and chronic obstructive pulmonary disease.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLE 1 GEP Binds to and Antagonizes TNFα Receptors (TNFR)

Modern methods of global analysis of protein-protein interactionsfollowed by biological assessment have led to powerful ways ofidentifying novel proteins not previously associated with thepathogenesis of a particular disease or organ system. Through afunctional genetic screen, we have now discovered that GEP, a novelmediator in chondrogenesis and arthritis, associates with TNFR. Thisextends our understanding of the action of growth factors and cytokinesin cartilage biology and their application to treatment of cartilagedisorders and arthritic conditions. In particular, our studies shedlight on a naturally occurring antagonist of the central proinflammatorycytokine TNFα, and provide insights into the degradative events thatoccur in patients with arthritic disorders. The identification andmanipulation of growth factors that regulate the chondrogenic potentialof mesenchymal stem cells and act as an inhibitors of a centralproinflammatory cytokine can be used to optimize the therapeuticapplication in cartilage disorders and connective tissue disorders.Important long-term goals of this work are (1) define the role of GEP,TNFα, as well as interaction and function interplay among them inregulating skeletal biology and related diseases; and (2) to recruitGEP, specially GEP-derived peptides, to develop new anti-TNF/TNFRtherapeutic interventions for various kinds of TNF-related diseases,including arthritis.

Our global screens led to the isolation of several novel GEP-bindingpartners and among them TNF receptors (TNFR) are of great interest tous. Subsequent studies showed that GEP directly bound to theextracellular domains of TNFR, and GEP-stimulated signaling and targetgene expression in chondrocytes strictly depends on TNFR. The fact thatboth GEP and TNF

bind to TNFR raised the possibility that the binding of GEP to TNFR mayblock the association of TNF

and its receptors, i.e., GEP may act as a naturally-occurring antagonistof TNFα. Indeed, GEP dramatically inhibits TNFα-induced inflammationresponse and chondrocyte apoptosis.

TNFR2 Identified as a GEP-Associated Receptor:

Taking into account the biological properties of GEP, it has beenhypothesized that GEP could act through “classic” membrane receptor(s),as do other known growth factors. Thus far, a functional receptor hasnot been identified. In a search for GEP-associated proteins we screeneda yeast two-hybrid (Y2H) cDNA library using the construct pDBleu-GEP(a.a.21-588) encoding GEP lacking signal peptide as bait, and isolated24 positive clones. Sequencing data from these clones showed that two ofthem were cell surface TNFR2 (TNFRSF1B/CD120b; Accession #NM_(—)130426).

GEP Binds to TNFR2 in Yeast and in Chondrocytes:

To verify the interaction between GEP and TNFR2 in yeast the plasmidencoding the GEP linked to Gal4 DBD and the plasmid encoding anN-terminal truncated mutant of TNFR2 (a.a. 26-567) fused to the VP16ADwere co-transformed into the yeast cells. Like the c-Jun/c-Fos pair,which is known to interact and used as a positive control, our assaysindicated that COMP interacts with GEP in yeast, based on the activityof β-galactosidase (FIG. 2A). To determine whether these two proteinsinteracted in primary human chondrocytes, a coimmunoprecipitation(Co-IP) assay was performed (FIG. 2B). Briefly, the cell extractsprepared from isolated human chondrocytes were incubated with eitheranti-TNFR2 antibody or control IgG, and the immunoprecipitated complexeswere subjected to a reducing SDS-PAGE and detected with anti-GEPantibodies. A specific GEP band was present in the immunoprecipitatedcomplexes brought down by anti-TNFR2 (lane 3), but not by control IgG(lane 2) antibodies, demonstrating that GEP specifically binds to theTNFR2 in primary human chondrocytes.

Direct Binding of GEP to the Extracellular Domains of TNFR1 and TNFR2:

Since there is remarkable amino acid similarity between extracellulardomains of TNFR1 and TNFR2, we next determined whether GEP directlybinds to TNFR1 and TNFR2 using solid-phase binding assay withrecombinant GEP and extracellular domains of TNFR1 and TNFR2 (R & DSystem) (FIG. 3). Briefly, microtiter plates were coated with 500 ng ofpurified GEP in 100 μl of TBS buffer (50 mM Tris/HCl, 150 mM NaCl,pH7.4). After blocking, various amounts (5-500 ng) of extracellulardomain of TNFR1 (TNFR1ECD, left panel) or extracellular domain of TNFR2(TNFR2ECD, right panel) were added to each well, and bound protein fromthe liquid phase was detected by antibody against TNFR1 or TNFR2,followed by a secondary antibody conjugated with horseradish peroxidase.As shown in FIG. 3, GEP demonstrated dose-dependent binding andsaturation to the liquid-phase TNFR1ECD and TNFR2ECD.

Cysteine-Rich Domain (CRD) of TNFR2 is Sufficient for Binding to GEP:

Various deletion mutants of TNFR2 were generated and tested in yeasttwo-hybrid assay for their ability to interact with GEP. Results fromfilter-based β-galactosidase assays (FIG. 4) of all these mutants aresummarized in FIG. 4A. Our conclusion from this set of experiments isthat each CRD (i.e. CRD1, CRD2, CRD3 or CRD4) of TNFR2 is sufficient forits interaction with GEP.

Anti-TNFR specific blocking antibodies or recombinant extracellulardomains of TNFR abolishes GEP-stimulated chondrocyte proliferation: Thefindings that GEP binds to the TNRF, together with our recent reportthat GEP has potent mitogenic effects on human chondrocytes [49], led usto determine whether GEP-stimulated chondrocyte proliferation depends onthe TNFR. Human chondrocytes were cultured in the absence (CTR) orpresence of 50 ng/ml GEP (GEP) or GEP plus either 1 ug/ml of anti-TNFR1(GEP+TNFR1 ab; SC-7895 is against extracellular domain of TNFR1),anti-TNFR2 (GEP+TNFR2 ab; SC-12751 is against extracellular domain ofTNFR2) or 1 ug/ml of anti-IGF1R (GEP+TGF1R ab, employed as a control),and cell proliferation was analyzed using an MTT assay (FIG. 5 leftpanel). As expected, GEP potently stimulated chondrocyte proliferation,and this GEP-stimulated cell proliferation was largely blocked by eitheranti-TNFR1 or anti-TNFR2 antibody whereas anti-IGF1R did not demonstrateany blocking effects on GEP action.

Since GEP directly associates with the extracellular domains of TNFR, wenext examined whether recombinant extracellular domains of TNFR willaffect GEP action in chondrocyte proliferation via competing withendogenous TNFR for interacting with GEP. As shown in the right panel ofFIG. 5, chondrocyte proliferation induced by 50 ng/ml GEP was completelyabolished by recombinant extracellular domain of either TNFR1 (R1ECD, 25ng/ml) or TNFR2 (R2ECD, 25 ng/ml). Taken together, these resultsindicate that GEP-mediated chondrocyte proliferation strictly depends onthe TNFR activity.

GEP Activates Akt and Erk1/2 Pathways in Chondrocytes:

We next sought to analyze GEP-activated signaling in chondrocytes usingThe PathScan® Multiplex Western Cocktail I (Cell Signaling) that allowsus to simultaneously detect levels of phospho-p90RSK, phospho-Akt,phosphop44/42 MAPK (Erk1/2) and phospho-S6 ribosomal protein on a singlemembrane. Human C28I2 chondrocytes (provided by Dr. Mary B. Goldring)were starved for 24 hours and treated with 50 ng/ml of GEP for varioustime points and cell lysates were analyzed using The PathScan® MultiplexWestern Cocktail I. As shown in FIG. 6, GEP specifically activated Aktand p44/p42 (Erk1/2) pathways in chondrocytes.

GEP-Mediated Activation of Target Genes Depends on TNFR:

To identify GEP downstream molecules, we performed genome-wide DNA chipanalysis. Total RNA was isolated from human C28I2 chondrocytes treatedwith 50 ng/ml of GEP for various time points and analyzed by microarrayanalysis (Affymetrix, Santa Clara, Calif.). Approximately 40 genes weredetermined to be upregulated (over 2-fold) following GEP treatment asdetermined by hierarchical clustering [53]. Interestingly, theGEP-inducible genes, including Gadd45β, JunB, KLF2, Samd7, Sox4 andTcf8, are also known to be activated by the TGF

subfamily [54-56]. We next determined whether activation of these genesby GEP depends on TNFR, expression profiling of these genes wereexamined using real-time PCR with primary wildtype (B6), TNFR1−/−, andTNFR2−/− MLE cells cultured in presence of 50 ng/ml GEP for various timepoints (FIG. 7). GEP clearly activated these genes in wildtype MLEcells; however, GEP largely lost these inductions in either TNFR1−/− orTNFR2−/− cells, indicating that GEP induction of its target genesdepends on TNFR1 and TNFR2. Interestingly, both TNFR1 and TNFR2 appearto be important for GEP-stimulated gene expression.

A proposed model for illustrating the TNFa- and GEP-inducedIntracellular events: GEP binds to the CRD of TNFR (FIG. 4), as doesTNFα [57]. Thus there exists a reciprocal inhibition of binding to TNFRbetween GEP and TNFα. An intriguing question is why GEP and TNFα, thatuse the same receptor TNFR, induce opposite responses. As illustrated inFIG. 8, TNF

trimmer binds to the extracellular domains of TNF receptors andinduces 1) a strong activation of the stress-related JNK and moderateresponse of the p38, and 2) activation of NF-kB pathway [58], whereasGEP potently activates Erk1/2 and moderately Akt signaling (FIG. 6 andFIG. 8). A number of GEP-activated genes, including Sox4, Smad7, JunB,Gadd45β, Tcf8, are also activated by the TGFβ subfamily, andGEP-mediated gene activation depends on TNFR (FIG. 7).

EXAMPLE 2 Discovery of Atsttrin (Antagonist of TNF/TNFR Signaling viaTargeting TNF Receptors)

To identify the peptide(s) of GEP required for binding to TNF receptors,a series of GEP mutants (i.e. C-terminal deletions, N-terminaldeletions, individual granulin unit, individual linker, as well asvarious combinations) were expressed in a yeast expression plasmid.Briefly, cDNA segments encoding the series of GEP mutants were amplifiedby PCR and cloned in-frame into the SalI/NotI sites of pDBleu (LifeTechnologies) yeast expression vector. The generated plasmids andpPC86-TNFR encoding extracellular domain of TNFR1/R2 was cotransformedinto the yeast MaV203 strain containing three reporter genes, His⁺,Ura⁺, and LacZ (Life Technologies), and transformants was examined forβ-galactosidase. Results from filter-based β-galactosidase assays (rightpanel in figures FIG. 9 through FIG. 15) of all these mutants aresummarized in the left panel of these figures. As revealed in FIG. 9,results from a series of C-terminal deletions indicated that deletionsfrom the C-terminal of GEP reduced the binding affinity to TNFR andfinally totally lost binding activity. This is also true for series ofN-terminal deletion mutants (FIG. 10).

Neither a single granulin unit (A, B, C, D, E, F, or G; FIG. 11) nor asingle linker unit (P1, P2, P3, P4, P5, P6 or P7; FIG. 12) could bind toTNFR, suggesting that the binding region of GEP may span one or moregranulin unit and linker. To examine this hypothesis, we first linkedeach granulin unit with its 3′-linker and found that granulin unit Fplus linker P3 exhibited weak binding to TNFR (FIG. 13). Next, we linkedeach granulin unit with its 5′-linker and found that P4 plus granulinunit A showed weak binding to TNFR, and also that P5 plus unit C showedweak binding to TNFR (FIG. 14). These findings led us to test thebinding of various combinations of half unit of A, C and F plus P3, P4and P5 to TNFR. As revealed in FIG. 15, ½A+P3+P4+½C+P5+½F demonstratedstrong binding to TNFR. This GEP derived peptide is now referred to asAtsttrin.

EXAMPLE 3 GEP Antagonizes TNF Action

Atsttrin antagonizes TNFα-induced inflammation response: We nextdetermined whether Atsttrin inhibits TNF-mediated inflammation.Neutrophils are triggered by inflammatory stimuli like TNF

to generate large quantities of reactive oxygen species that contributeto neutrophil activation and the development of inflammatory processes[59]. Accordingly, we tested the effect of Atsttrin on TNFα-inducedneutrophil activation. As shown in FIG. 16A, Atsttrin dose-dependentlyinhibits neutrophil activation triggered by TNFα. Importantly andremarkably, Atsttrin exhibits inhibition that rivals (is at least asgood as) Enbrel and Remicade (FIG. 16B), which have been used clinicallyfor treating various kinds of inflammatory diseases, includingparticularly rheumatoid arthritis [60].

GEP inhibits TNFα-induced cell death: TNF-α has been shown to induceapoptosis in chondrocyte cultures [61-63]. We next determined whetherGEP affected TNF-α-induced apoptosis in chondrocytes. Briefly, ratchondrosarcoma (RCS) cells and human C28I2 chondrocytes wereserum-starved for 24 h to remove the effect of exogenous growth factorsand cytokines. Thereafter, cells were stimulated with 0.02% BSA (CTR,control), 100 ng/ml of GEP (GEP), 100 ng/ml of TNF-α, or GEP plus TNF-αfor 36 h. Apoptosis was measured by using an TUNEL assay kit (Promega).As shown in FIG. 17, TNF-α induced prominent cell death in both RCS andC28I2 chondrocytes, whereas GEP dramatically inhibited TNF-α inductionof apoptosis in chondrocytes.

GEP inhibits TNFα-induced metalloproteinase: We have recently reportedthat TNFα induces expression of ADAMTS-7 and ADAMTS-12, twometalloproteinases in the ADAMTS family [64]. We next determined whetherGEP inhibited induction of ADAMTS by TNFα. Briefly, human C28I2chondrocytes or cartilage explants were treated with TNF

in the absence or presence of various amount of GEP for 48 hours, andthe expression of ADAMTS-7 and ADAMTS-12 were determined using real-timePCR with their specific primers. In accordance with our previouslyreport results [64], TNF

clearly induced ADAMTS-7 and ADAMTS-12 expression (FIG. 18). GEPdemonstrated a dose-dependent inhibition of TNFα-mediated induction ofmetalloproteinase in chondrocytes or cartilage explants (FIG. 18).

Brief Summary of Major Characteristics of GEP and Atsttrin: Compared tothe currently available anti-inflammation engineered antibodies orrecombinant protein blockers, including etanercept (Enbrel, a solubleTNFR2-IgG1 fusion protein), infliximab (Remicade, a chimeric monoclonalantibody against TNF-α), and adalimumab (a humaneric monoclonal antibodyagainst TNF-α), GEP and its derived Atsttrin have the features as below:

i) Unique anti-TNF/TNFR property: Antibodies and immunoadhesins thatdirectly target cytokines for their systemic removal (ligand ablation)have become an effective therapeutic strategy (e.g. etanercept,adalimumab and infliximab), and in some indications the selectivetargeting of cytokine receptors (e.g. anakinra) can deliver a morehighly effective clinical outcome. Our studies demonstrates that GEP isthe first known growth factor that directly targets to TNF receptors(TNFR), thus GEP and its derived peptide(s) represent the firstanti-TNF/TNFR signaling blockers through acting on the cytokinereceptors, resembling the action of Anakinra that targets to IL-1receptor. (See FIG. 19 for a comparison of the distinctanti-inflammation mechanisms between GEP/Atsttrin and Enbrel/Remicade).Due to its unique anti-TNF/TNFR signaling activity, Atsttrin may alsowork well for the patient population who does not respond to currentTNFα blockers, such as Remicade.

ii) Low toxicity: Since Atsttrin is derived a naturally-occurring GEPfactor that is already present in body fluids, it is expected that GEPand Atsttrin will not cause immunologic issues or responses and willexhibit no or less toxicity when compared to other engineeredrecombinant proteins.

iii) Multiple Functions: In addition to its anti-inflammatory activity,GEP also has potent tissue-repair function, thus GEP and Atsttrin areexpected to block inflammation reaction on one hand, and also repair theinjured tissues by inflammation response. In contrast, all currentanti-TNF blockers, including Enbrel and Remicade, do not havetissue-repair activity. Furthermore, our pilot studies indicated thatAtsttrin has tumor-suppression activity, which represents anotherapplication of Atsttrin.

EXAMPLE 4 Atsttrin Inhibits GEP-Stimulated Cancer Cell Proliferation

High levels of GEP expression are found in several human cancers andcontribute to tumorigenesis in diverse cancers, including breast cancer,clear cell renal carcinoma, invasive ovarian carcinoma, glioblastoma,adipocytic teratoma, and multiple myeloma [16, 18-24]. Atsttrin inhibitsTNF-mediated inflammation via blocking the binding of TNF to TNFR, andit is expected that Atsttrin may also inhibit tumor cell growth viablocking the binding of GEP to TNFR. To test this hypothesis we examinedthe effects of Atsttrin on GEP-stimulated cell proliferation of cancercells. As revealed in FIG. 20, Atsttrin dose-dependently inhibitedGEP-stimulated cell proliferation of cancer cells tested.

References

-   1. Martel-Pelletier, J., Pathophysiology of osteoarthritis.    Osteoarthritis Cartilage, 1999. 7(4): p. 371-3.-   2. Petersson, I. F., et al., Changes in cartilage and bone    metabolism identified by serum markers in early osteoarthritis of    the knee joint. Br J Rheumatol, 1998. 37(1): p. 46-50.-   3. Ayral, X., Diagnostic and quantitative arthroscopy: quantitative    arthroscopy. Baillieres Clin Rheumatol, 1996. 10(3): p. 477-94.-   4. Ayral, X., et al., Effects of video information on preoperative    anxiety level and tolerability of joint lavage in knee    osteoarthritis. Arthritis Rheum, 2002. 47(4): p. 380-2.-   5. Aigner, T., et al., Reexpression of type IIA procollagen by adult    articular chondrocytes in osteoarthritic cartilage. Arthritis    Rheum, 1999. 42(7): p. 1443-50.-   6. Lippiello, L., D. Hall, and H. J. Mankin, Collagen synthesis in    normal and osteoarthritic human cartilage. J Clin Invest, 1977.    59(4): p. 593-600.-   7. Sandell, L. J. and T. Aigner, Articular cartilage and changes in    arthritis. An introduction: cell biology of osteoarthritis.    Arthritis Res, 2001. 3(2): p. 107-13.-   8. Wright, W. E., D. A. Sassoon, and V. K. Lin, Myogenin, a factor    regulating myogenesis, has a domain homologous to MyoD. Cell, 1989.    56(4): p. 607-17.-   9. Zhou, J., et al., Purification of an autocrine growth factor    homologous with mouse epithelin precursor from a highly tumorigenic    cell line. J Biol Chem, 1993. 268(15): p. 10863-9.-   10. Anakwe, O. O. and G. L. Gerton, Acrosome biogenesis begins    during meiosis: evidence from the synthesis and distribution of an    acrosomal glycoprotein, acrogranin, during guinea pig    spermatogenesis. Biol Reprod, 1990. 42(2): p. 317-28.-   11. Baba, T., et al., Acrogranin, an acrosomal cysteine-rich    glycoprotein, is the precursor of the growth modulating peptides,    granulins, and epithelins, and is expressed in somatic as well as    male germ cells. Mol Reprod Dev, 1993. 34(3): p. 233-43.-   12. Daniel, R., et al., Cellular localization of gene expression for    progranulin. J Histochem Cytochem, 2000. 48(7): p. 999-1009.-   13. Zanocco-Marani, T., et al., Biological activities and signaling    pathways of the granulin/epithelin precursor. Cancer Res, 1999.    59(20): p. 5331-40.-   14. Ong, C. H. and A. Bateman, Progranulin (granulin-epithelin    precursor, PC-cell derived growth factor, acrogranin) in    proliferation and tumorigenesis. Histol Histopathol, 2003. 18(4): p.    1275-88.-   15. Hrabal, R., et al., The hairpin stack fold, a novel protein    architecture for a new family of protein growth factors. Nat Struct    Biol, 1996. 3(9): p. 747-52.-   16. Davidson, B., et al., Granulin-epithelin precursor is a novel    prognostic marker in epithelial ovarian carcinoma. Cancer, 2004.    100(10): p. 2139-47.-   17. Lu, R. and G. Serrero, Inhibition of PC cell-derived growth    factor (PCDGF, epithelin/granulin precursor) expression by antisense    PCDGF cDNA transfection inhibits tumorigenicity of the human breast    carcinoma cell line MDA-MB-468. Proc Natl Acad Sci USA, 2000.    97(8): p. 3993-8.-   18. Bateman, A., et al., Granulins, a novel class of peptide from    leukocytes. Biochem Biophys Res Commun, 1990. 173(3): p. 1161-8.-   19. Gonzalez, E. M., et al., A novel interaction between perlecan    protein core and progranulin: potential effects on tumor growth. J    Biol Chem, 2003. 278(40): p. 38113-6.-   20. He, Z. and A. Bateman, Progranulin (granulin-epithelin    precursor, PC-cell-derived growth factor, acrogranin) mediates    tissue repair and tumorigenesis. J Mol Med, 2003. 81(10): p. 600-12.-   21. He, Z., et al., Progranulin is a mediator of the wound response.    Nat Med, 2003. 9(2): p. 225-9.-   22. Jones, M. B., M. Spooner, and E. C. Kohn, The granulin-epithelin    precursor: a putative new growth factor for ovarian cancer. Gynecol    Oncol, 2003. 88(1 Pt 2): p. S136-9.-   23. Wang, W., et al., PC cell-derived growth factor (granulin    precursor) expression and action in human multiple myeloma. Clin    Cancer Res, 2003. 9(6): p. 2221-8.-   24. Zhang, H. and G. Serrero, Inhibition of tumorigenicity of the    teratoma PC cell line by transfection with antisense cDNA for PC    cell-derived growth factor (PCDGF, epithelin/granulin precursor).    Proc Natl Acad Sci USA, 1998. 95(24): p. 14202-7.-   25. Hogue, M., et al., Granulin and granulin repeats interact with    the Tat.P-TEFb complex and inhibit Tat transactivation. J Biol    Chem, 2005. 280(14): p. 13648-57.-   26. Hogue, M., et al., The growth factor granulin interacts with    cyclin T1 and modulates P-TEFb-dependent transcription. Mol Cell    Biol, 2003. 23(5): p. 1688-702.-   27. Thornburg, N. J., S. Kusano, and N. Raab-Traub, Identification    of Epstein-Barr virus RK-BARF0-interacting proteins and    characterization of expression pattern. J Virol, 2004. 78(23): p.    12848-56.-   28. Sell, C., et al., Effect of a null mutation of the insulin-like    growth factor I receptor gene on growth and transformation of mouse    embryo fibroblasts. Mol Cell Biol, 1994. 14(6): p. 3604-12.-   29. Xu, S. Q., et al., The granulin/epithelin precursor abrogates    the requirement for the insulin-like growth factor 1 receptor for    growth in vitro. J Biol Chem, 1998.273(32): p. 20078-83.-   30. Sun, X., M. Gulyas, and A. Hjerpe, Mesothelial differentiation    as reflected by differential gene expression. Am J Respir Cell Mol    Biol, 2004. 30(4): p. 510-8.-   31. Suzuki, M. and M. Nishiahara, Granulin precursor gene: a sex    steroid-inducible gene involved in sexual differentiation of the rat    brain. Mol Genet Metab, 2002. 75(1): p. 31-7.-   32. Barreda, D. R., et al., Differentially expressed genes that    encode potential markers of goldfish macrophage development in    vitro. Dev Comp Immunol, 2004. 28(7-8): p. 727-46.-   33. Justen, H. P., et al., Differential gene expression in synovium    of rheumatoid arthritis and osteoarthritis. Mol Cell Biol Res    Commun, 2000. 3(3): p. 165-72.-   34. Zhu, J., et al., Conversion of proepithelin to epithelins: roles    of SLPI and elastase in host defense and wound repair. Cell, 2002.    111(6): p. 867-78.-   35. Baker, M., et al., Mutations in progranulin cause tau-negative    frontotemporal dementia linked to chromosome 17. Nature, 2006.    442(7105): p. 916-9.-   36. Cruts, M., et al., Null mutations in progranulin cause ubiquitin    positive frontotemporal dementia linked to chromosome 17q21.    Nature, 2006. 442(7105): p. 920-4.-   37. Gass, J., et al., Mutations in progranulin are a major cause of    ubiquitin positive frontotemporal lobar degeneration. Hum Mol    Genet, 2006. 15(20): p. 2988-3001.-   38. Rowland, L. P., Frontotemporal dementia, chromosome 17, and    progranulin. Ann Neurol, 2006. 60(3): p. 275-7.-   39. Arikawa-Hirasawa, E., et al., Perlecan is essential for    cartilage and cephalic development. Nat Genet, 1999. 23(3): p.    354-8.-   40. Kvist, A. J., et al., Chondroitin sulfate perlecan enhances    collagen fibril formation. Implications for perlecan    chondrodysplasias. J Biol Chem, 2006. 281(44): p. 33127-39.-   41. Nicole, S., et al., Perlecan, the major proteoglycan of basement    membranes, is altered in patients with Schwartz-Jampel syndrome    (chondrodystrophic myotonia). Nat Genet, 2000. 26(4): p. 480-3.-   42. Wallach, D., et al., Tumor necrosis factor receptor and Fas    signaling mechanisms. Annu Rev Immunol, 1999. 17: p. 331-67.-   43. Guicciardi, M. E. and G. J. Gores, AlP1: a new player in TNF    signaling. J Clin Invest, 2003. 111(12): p. 1813-5.-   44. Gupta, S., A decision between life and death during    TNF-alpha-induced signaling. J Clin Immunol, 2002. 22(4): p. 185-94.-   45. Kollias, G. and D. Kontoyiannis, Role of TNF/TNFR in    autoimmunity: specific TNF receptor blockade may be advantageous to    anti-TNF treatments. Cytokine Growth Factor Rev, 2002. 13(4-5): p.    315-21.-   46. MacRae, V. E., et al., Cytokine actions in growth disorders    associated with pediatric chronic inflammatory diseases (review).    Int J Mol Med, 2006. 18(6): p. 1011-8.-   47. MacRae, V. E., et al., Cytokine profiling and in vitro studies    of murine bone growth using biological fluids from children with    juvenile idiopathic arthritis. Clin Endocrinol (Oxf), 2007.    67(3): p. 442-8.-   48. Martensson, K., D. Chrysis, and L. Savendahl, Interleukin-1beta    and TNF-alpha act in synergy to inhibit longitudinal growth in fetal    rat metatarsal bones. J Bone Miner Res, 2004. 19(11): p. 1805-12.-   49. Xu, K., et al., Cartilage oligomeric matrix protein associates    with granulin-epithelin precursor (GEP) and potentiates    GEP-stimulated chondrocyte proliferation. J Biol Chem, 2007.    282(15): p. 11347-55.-   50. Johnson, K. A., et al., Vanin-1 pantetheinase drives increased    chondrogenic potential of mesenchymal precursors in ank/ank mice. Am    J Pathol, 2008. 172(2): p. 440-53.-   51. Meirelles Lda, S, and N. B. Nardi, Murine marrow-derived    mesenchymal stem cell: isolation, in vitro expansion, and    characterization. Br J Haematol, 2003. 123(4): p. 702-11.-   52. Longobardi, L., et al., Effect of IGF-I in the chondrogenesis of    bone marrow mesenchymal stem cells in the presence or absence of    TGF-beta signaling. J Bone Miner Res, 2006. 21(4): p. 626-36.-   53. Attur, M. G., et al., “A system biology” approach to    bioinformatics and functional genomics in complex human diseases:    arthritis. Curr Issues Mol Biol, 2002. 4(4): p. 129-46.-   54. Yang, Y. C., et al., Hierarchical model of gene regulation by    transforming growth factor beta. Proc Natl Acad Sci USA, 2003.    100(18): p. 10269-74.-   55. Zavadil, J., et al., Genetic programs of epithelial cell    plasticity directed by transforming growth factorbeta. Proc Natl    Acad Sci USA, 2001. 98(12): p. 6686-91.-   56. Zavadil, J., et al., Integration of TGF-beta/Smad and    Jagged1/Notch signalling in epithelial-tomesenchymal transition.    Embo J, 2004. 23(5): p. 1155-65.-   57. Ware, C. F., The TNF superfamily. Cytokine Growth Factor    Rev, 2003. 14(3-4): p. 181-4.-   58. Royuela, M., et al., TNF-alpha/IL-1/NF-kappaB transduction    pathway in human cancer prostate. Histol Histopathol, 2008.    23(10): p. 1279-90.-   59. Nathan, C. F., Neutrophil activation on biological surfaces.    Massive secretion of hydrogen peroxide in response to products of    macrophages and lymphocytes. J Clin Invest, 1987. 80(6): p. 1550-60.-   60. Rothe, A., B. E. Power, and P. J. Hudson, Therapeutic advances    in rheumatology with the use of recombinant proteins. Nat Clin Pract    Rheumatol, 2008. 4(11): p. 605-14.-   61. Aizawa, T., et al., Induction of apoptosis in chondrocytes by    tumor necrosis factor-alpha. J Orthop Res, 2001. 19(5): p. 785-96.-   62. Horiguchi, M., et al., Tumour necrosis factor-alpha up-regulates    the expression of BMP-4 mRNA but inhibits chondrogenesis in mouse    clonal chondrogenic EC cells, ATDC5. Cytokine, 2000. 12(5): p.    526-530.-   63. MacRae, V. E., C. Farquharson, and S. F. Ahmed, The    pathophysiology of the growth plate in juvenile idiopathic    arthritis. Rheumatology (Oxford), 2006. 45(1): p. 11-9.-   64. Luan, Y., et al., Inhibition of ADAMTS-7 and ADAMTS-12    degradation of cartilage oligomeric matrix protein by    alpha-2-macroglobulin. Osteoarthritis Cartilage, 2008. 16(11): p.    1413-20.-   65. He, Z. and Bateman, A., Progranulin gene expression regulates    epithelial cell growth and promotes tumor growth in vivo. Cancer    Res, 1999. 59, p. 3222.-   66. He, Z. et al., Progranulin is a mediator of the wound response.    Nat Med, 2003. 9, p. 225.-   67. Zhu, J. et al., Conversion of proepithelin to epithelins: roles    of SLPI and elastase in host defense and wound repair. Cell, 2002.    111, p. 867.

EXAMPLE 5 Acute Inflammation Air Pouch Model Studies

Anti-inflammatory activity of GEP and Atsttrin were tested in an airpouch-induced acute inflammation animal model. To induce air pouches,10-15-week-old male mice were injected subcutaneously on the back with 3ml of air. After 2 days, the pouches were reinflated with 1.5 ml of air.On day 6, inflammation was induced by injection of 1 ml of a suspensionof carrageenan (2% weight/volume in calcium- and magnesium-freephosphate buffered saline solution [PBS]) into the air pouch. Remicade(10 ug/g), GEP (10 ug/g) and Atsttrin (10 ug/g) was administered 1 hourprior to induction of inflammation in the air pouch. After 4 hours, themice were killed by CO, narcosis, the pouches were flushed with 2 ml ofPBS, and exudates were harvested. After centrifugation (1,000 g for 10minutes), the cell-free exudates were collected. The IL-6 and IL-13concentration was quantitated in the exudates in duplicate byenzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn.)following the manufacturer's instructions. As showed in FIG. 21, bothGEP and its derived Atsttrin potently reduce the level of IL-6 andIL-13, two major inflammatory mediators. Note that both GEP and Atsttrinexhibit more effective anti-inflammation (approximately 80% reduction inIL-13 concentration) than Remicade in this model.

EXAMPLE 6 Chronic Inflammation Animal Model Studies

GEP activity is determined in a rheumatoid arthritis animal model. Inthis study, female BALB/cJ mice are used. The animals are housed at adensity of three to four per cage and allowed to acclimate for one week.A combination of four different monoclonal antibodies to thewell-defined epitopes of Collagen II (mAbs: C11b, J1, D3, and U1) areadministered IV (on Day 0). Three days later (Day 3), mice arechallenged with lipopolysaccharide administered IP (LPS, 25 μg/mouse).Test compound and vehicle are administered according to study designbeginning one hour post-LPS challenge on Day 3.Paw edema is measured onDays −1, 4, 7, 10 and 11. Arthritis Scores in mice are visually examinedfor signs of joint inflammation on Days −1, 3, 5, 7, and 10. Bodyweights are determined on all days when arthritis scores are obtained.Blood is drawn at necropsy and processed to serum or plasma. Mice areeuthanized after obtaining final caliper measurements on Day 11. Hindlegs are taken and fixed for histopathology analysis.

EXAMPLE 7 GEP and Atsttrin Effects on Progression of Arthritis in TNFαTransgenic Mice

Mice transgenic for human TNFα, originally generated by Dr. GeorgeKollias' laboratory, develop a chronic inflammatory and destructivepolyarthritis with many characteristics observed in rheumatoid arthritispatients [55]. The phenotype of this mouse model validated the theorythat TNFα is at the apex of the pro-inflammatory cascade in rheumatoidarthritis, and foreshadowed the remarkable success of anti-TNFα therapythat has transformed the effective management of this disease. As such,the TNFα transgenic mice are very useful tools for dissecting themolecular mechanisms of the pathogenic process and evaluating theefficacy of novel therapeutic strategies for rheumatoid arthritis [115,138, 139]. This model is used to further assess GEP, especially itsderived peptide(s) atsttrin, direct administration to treat rheumatoidarthritis. Briefly, TNFα transgenic mice (n=60, purchased from Taconic)are divided into 6 groups of 10 mice each and receive GEP, GEP-derivedpeptide, and anti-TNFa antibody (serves as a control) at doses describedin the literature as being effective [140, 141]. All treatments areadministered by intraperitoneal injection. Group 1 is treated withphosphate buffered saline and serves as a negative control. Group 2 and3 (low does of GEP and GEP-derived peptide(s)) receive 1 mg/g of GEP orpeptide(s) 3 times weekly from week 4 to week 10. Group 4 and 5 (highdoes) receive 10 mg/g of GEP or peptide(s) 3 times weekly from week 4 toweek 10. Group 6 receive 10 mg/g of anti-TNFα 3 times weekly from week 4to week 10. At week 10, all animals are sacrificed by cervicaldislocation, blood is withdrawn by heart puncture, and the paws andtibial bones are dislocated for further analyses. Clinical evaluation isperformed weekly, starting at 4 weeks after birth. Arthritis isevaluated in each group of animals.

EXAMPLE 8 GEP and Atsttrin Effects on RANKL-Induced Osteoclastogenesis

Osteoclasts are the bone-resorbing cells, and their excess activitycauses osteoporosis. RANKL is the key receptor for osteoclastogenesisand binds RANK. RANK and TNFR belong to the same TNFR family and theyshare significant similarity in sequence and particularly in structure.Given that (1) RANK, the key receptor for osteoclastogenesis, belongs tothe TNFR subfamily, and that (2) GEP and its derived peptide Atsttrinbinds to TNFR and blocks TNF alpha action, we also examined whether GEPand Atsttrin affect osteoclastogenesis. RANKL-induced osteoclastogenesiswas assessed in the presence and absence of GEP or peptide atsttrin.Briefly, we cultured Raw 264.7 macrophages in the presence of RANKL for4 days and TRAP staining was performed. As expected, RANKL inducedrobust osteoclastogenesis and TRAP multinucleated positive cells wereobserved (FIG. 22, indicated with arrows). GEP and atsttrin demonstrateddoes-dependent inhibition of osteoblast differentiation (FIG. 22).Interesting, atsttrin appears to be more potent than GEP in blockingosteoclastogenesis. The finding that atsttrin inhibitsosteoclastogenesis indicates that this peptide also has potential fortreating osteoporosis in addition to various kinds of inflammatorydiseases, including rheumatoid arthritis.

EXAMPLE 9 GEP and Atsttrin Binding to TNF Family Members RANKL and FAS

The interactions between GEP/Atsttrin and other members in TNF receptors(TNFR) subfamily, including RANK and FAS, were examined. To assessbinding, various pairs of plasmids, were co-transformed into yeaststrain MAV203 and a yeast two-hybrid assay was performed (FIG. 26). GEPplasmids were co-transformed with each of TNFR1, TNFR2, RANK and FASplasmids. Atsttrin plasmids were co-transformed with each of TNFR1,TNFR2, RANK and FAS plasmids. Yeast transformants were selected onSD-leu⁻/trp⁻/his⁻/ura⁻/3AT⁺ plates and tested for β-galactosidaseactivity. The lack of interaction between Rb and lamin was used as anegative control. By this two-hybrid assay, GEP associates with RANK andFAS in addition to TNFR, whereas Atsttrin specifically binds to TNFR.

These studies demonstrate that GEP also binds to RANK and Fas, althoughthe interaction may be weaker than that with TNFR1 and TNFR2. Incontrast, Atsttrin, which shows higher binding affinity to TNF (TNFR1and TNFR2) receptors than does GEP, does not interact directly with RANKand FAS as assessed by two-hybrid binding assay. Thus, due to itsspecificity for TNFR, Atsttrin may have less or distinct side-effectsand toxicity than does GEP because GEP associates with the other memberof the TNFR family RANK and thus may affect multiple pathophysiologicalprocesses.

We have previously showed that Atsttrin potently inhibits RANKL-inducedosteoclastogenesis (Example 8). The present example results nowdemonstrate that Atsttrin does not bind to RANK. Collectively, thesedata suggest that Atsttrin-mediated inhibition of osteoclastgenesis mustbe through blocking the TNF/TNFR pathway. Indeed, growing evidencedemonstrates that TNF/TNFR signaling is also crucial forosteoclastogenesis.

EXAMPLE 10 GEP Exhibits Higher Binding Affinity for TNFR Than Does TNFα

Kinetic binding studies of GEP and TNFα to TNFR were observed andanalyzed using Analytical Surface Plasmon Resonance with SensiQ Pioneer(ICx Nomadics, Oklahoma City, Okla. 73104). The kinetic binding is shownin FIG. 27, and the kinetic constants are summarized in TABLE 1.Remarkably, GEP exhibits higher affinity for TNF receptors, especiallyTNFR2 when compared to TNFα (K_(D) of GEP vs. TNFα: 1.28×10⁻⁹M vs.7.64×10⁻⁷M). In contrast to TNFα, which shows much higher affinity forTNFR1 (KD 8.8×10⁻⁹ M) than TNFR2 (KD 7.64×10⁻⁷M), GEP exhibits slightlyhigher affinity for TNFR2 (KD 1.28×10⁻⁹ M) than TNFR1 (KD 1.58×10⁻⁹M).

TABLE 1 Kinetic Constants Analyte K_(a) (×10⁴ M⁻¹S⁻¹) K_(d) (×10⁻⁴ S⁻¹)K_(D) (M) TNFα/TNFR1 8.25 7.27 8.80 × 10⁻⁹ GEP/TNFR1 51.6 8.14 1.58 ×10⁻⁹ TNFα/TNFR2 7.79 59.5 7.64 × 10⁻⁷ GEP/TNFR2 540 6.92 1.28 × 10⁻⁹

EXAMPLE 11 Binding Studies of Atsttrin and TNFR

Atsttrin was expressed in bacteria as a GST fusion protein, purified onglutathione agarose resin, and eluted using Xa factor (there is a Xafactor cleavage site between GST and Atsttrin) (FIG. 28A). Xa factor wasthen removed from the elution using Xa Removal Resin (Qiagen). PurifiedAtsttrin was analyzed for endotoxin using the Limulus amebocyte lysateassay, which indicated endotoxin levels similar to control medium, manyfold below the manufacturer's specification of <1 unit/μg. UsingAnalytical Surface Plasmon Resonance Assay (FIG. 28 B & C), we wereexcited to find that Atsttrin exhibited ˜9-fold higher binding affinityfor TNFR2 (KD of Atsttrin/TNFR2 vs. TNFα/TNFR2: 8.59×10⁻⁸M vs.7.64×10⁻⁷M), but ˜18-fold lower affinity for TNFR1 than TNFα (KD ofAtsttrin/TNFR1 vs. TNFα/TNFR1: 1.60×10⁻⁷M vs. 8.80×10⁻⁹M), suggestingthat Atsttrin can block the TNFα/TNFR2 pathway effectively, but may notsignificantly affect TNFα/TNFR1 signaling.

EXAMPLE 12 Atsttrin Fails to Activate Erk1/2 and Alit Signaling andInhibits Cancer Cell Proliferation

Using the PathScan® Multiplex Western Cocktail I (Cell Signaling) thatallows one to simultaneously detect levels of phospho-p90RSK,phospho-Akt, phospho-p44/42 MAPK (Erk1/2), and phospho-S6 ribosomalprotein on a single membrane, we next sought to compare GEP- andAtsttrin-activated signaling in chondrocytes. Human C28I2 chondrocytes(provided by Dr. Mary B. Goldring) were starved for 24 hr and treatedwith 50 ng/ml of GEP or Atsttrin for various time points, and celllysates were analyzed using the PathScan® Multiplex Western Cocktail I.As shown in FIG. 29, GEP strongly activated Erk1/2 and moderatelyactivated Akt pathways (Feng, J., Guo, F., Jiang, B., Frenkel, S.,Zhang, Y., Wang, D., Liu, C. J., GEP: A BMP2-Inducible Growth Factorthat Activates Erk1/2 Signaling and JunB Transcription Factor inChondrogenesis, FASEB J., 2010 Feb. 2 [Epub ahead of print]). However,Atsttrin, while retaining the TNFR-binding activity of GEP (FIG. 28),loses GEP's oncogenic signaling. When GEP and Atsttrin are tested incombination, Atsttrin blocks or suppresses GEP-mediated activation ofp-AKT and p-ERK (FIG. 29).

EXAMPLE 13 Atsttrin Prevents the Onset of Arthritis in aCollagen-Induced arthritis (CIA) Model

We next examined Atsttrin in a collagen-induced arthritis (CIA) mousemodel that exhibits many of the clinical and pathological features ofRA. Briefly, DBA/1 mice were challenged on day 0 with chick collagen IIemulsified in modified complete Freund's adjuvant given s.c. at the baseof the tail (Chondrex Single Immunization). On day 19, mice were dividedinto three groups (each n=10) and treated every other day until day 35,as follows: Group 1 received Atsttrin at a dose of 10 pg/g body weighti.p., Group 2 received Enbrel (soluble extracellular domain of TNFR2) ata dose of 10 pg/g body weight i.p. (serving as positive control), andGroup 3 received an equal volume of phosphate-buffered saline (PBS,serving as negative control). Mice were monitored daily for incidence ofarthritis, arthritis severity score, and paw thickness measured by aconstant pressure caliper. As shown in FIGS. 30A and 30B, both Atsttrinand Enbrel effectively prevented the development of arthritis. Atsttrinwas more potent than Enbrel in this model, since Atsttrin completelyprevented the onset of arthritis. As seen in the representative panelsin FIG. 31, symptoms of CIA (swelling, erythema, deformity) wereapparent in mice treated with PBS. In contrast, mice treated withAtsttrin and Enbrel demonstrated markedly reduced pathology, andAtsttrin-treated mice were similar to normal mice. Ankles from CIA micetreated with PBS exhibited robust leukocyte infiltration and tissuedestruction (H&E staining) and loss of matrix staining (Sarfranin-Ostaining) (FIG. 32A). Arthritic symptoms were absent in Atsttrin-treatedmice. MicroCT images (FIG. 32B) revealed clear bone erosion in CIA micetreated with PBS, but not with Atsttrin. In addition, bone-resorbingosteoclasts were clearly seen with TRAP+ around the erosive area in CIAmice treated with PBS; in contrast, TRAP+ osteoclasts were hardlydetectable in Atsttrin-treated CIA mice (FIG. 33).

We evaluated Atsttrin effects of the levels of proinflammatory andanti-inflammatory cytokines in the sera of the CIA mice. Atsttrin wascompared to PBS and Enbrel in these studies. Proinflammatory cytokinesIL-1β and IL-6 were evaluated and anti-inflammatory cytokines IL-10 andIL-13 were assessed. As shown in FIG. 34, both Atsttrin and Enbrelsignificantly reduced the levels of the pro-inflammatory cytokine IL-6.IL-1β was less significantly reduced by Enbrel and Atsttrin, althoughAtsttrin reduced IL-1β more than Enbrel did. With regard toanti-inflammatory cytokines, while both Enbrel and Atsttrin increasedIL-10, Atsttrin showed a more significant effect on IL-10 levels. BothEnbrel and Atsttrin increased the amount of the anti-inflammatorycytokine IL-13.

EXAMPLE 14 Effects of Atsttrin on TNF-Induced Activities in Cells

In vitro cell-based studies in RAW 264.7 cells were utilized to furtherassess and evaluate Atsttrin effects of TNF responses in cells. GEP andAtsttrin were evaluated for their effects on TNF-induced nitriteproduction. Nitrite production was determined in cells in the presenceof added TNF (500 ng/ml) and with increasing amounts of either GEP,Atsttrin or Enbrel (0.3, 1.5 and 7.5 nM). While GEP reduced nitriteproduction somewhat, each of Atsttrin and Enrel reduced TNF-inducednitrite production more significantly (FIG. 36). Next, TNF-inducednuclear accumulation of NFκB was evaluate in cells. Both GEP andAtsttrin blocked TNF-induced nuclear accumulation of NFκB as assessed byNFκB p65 staining (FIG. 37). Induction of a TNF-activated NFκB reportergene (PAK-1 with an NFκB binding site) was determined in the presence ofTNFα and either GEP or Atattrin at increasing concentrations (FIG. 38).Atsttrin inhibited the reporter gene's induction more significantly thatGEP at lower concentrations.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1. An isolated peptide comprising Granulin/epithelin precursor (GEP)granulin units F (amino acids 126-178 of SEQ ID NO: 1), A (amino acids284-335 of SEQ ID NO: 1), and C (amino acids 366-416 of SEQ ID NO: 1);and GEP linker units P3 (amino acids 179-205 of SEQ ID NO:1), P4 (aminoacids 262-283 of SEQ ID NO:1), and P5 (amino acids 336-365 of SEQ IDNO:1), wherein the peptide antagonizes TNFR1 or TNFR2 signaling, andwherein the peptide is a mixed portion of the full-length GEP sequence.2. An isolated peptide comprising: at least ½F (amino acids 153-178 ofSEQ ID NO: 1); P3 (amino acids 179-205 of SEQ ID NO:1) P4 (amino acids262-283 of SEQ ID NO:1) at least ½A (amino acids 284-304 of SEQ ID NO:1); P5 (amino acids 336-365 of SEQ ID NO:1); and at least ½C (aminoacids 366-392 of SEQ ID NO: 1); wherein the peptide antagonizes TNFR1 orTNFR2 signaling, and wherein the peptide is a mixed portion of thefull-length GEP sequence.
 3. The isolated peptide of claim 1,comprising: (a) amino acids 126-205 of SEQ ID NO: 1; (b) amino acids262-335 of SEQ ID NO: 1; and (c) amino acids 336-416 of SEQ ID NO:
 1. 4.An isolated peptide comprising SEQ ID NO: 2 or a variant thereof havingat least 90% sequence identity to SEQ ID NO: 2, wherein the peptideantagonizes TNFR1 or TNFR2 signaling.
 5. The isolated peptide of claim4, wherein the peptide comprises SEQ ID NO:
 2. 6. A pharmaceuticalcomposition comprising the isolated peptide of claim 1 and apharmaceutically acceptable carrier, vehicle, or diluent.
 7. Thecomposition of claim 6 further comprising one or more of ananti-inflammatory agent or compound, an anti-cancer agent or compound,and an immunomodulatory agent.
 8. A pharmaceutical compositioncomprising the isolated peptide of claim 4 and a pharmaceuticallyacceptable carrier, vehicle, or diluent.
 9. The composition of claim 8further comprising one or more of an anti-inflammatory agent orcompound, an anti-cancer agent or compound, and an immunomodulatoryagent.
 10. A pharmaceutical composition comprising the isolated peptideof claim 2 and a pharmaceutically acceptable carrier, vehicle, ordiluent.
 11. The composition of claim 10 further comprising one or moreof an anti-inflammatory agent or compound, an anti-cancer agent orcompound, and an immunomodulatory agent.